Benzene-derived free radical--a possible mediator of its carcinogenicity. I. Ultrastructural and functional studies on the effect of benzene on human white blood cells and its possible prevention.

The assumption that benzene exerts its leukemogenic action on cell components after being converted to a transient and relatively stable free radical is supported by the in vitro effect of benzene on the ultrastructure of human peripheral blood polymorphonuclear (PMN) cells, lymphocytes and monocytes. The effect was examined with a transmission and scanning electron microscope and was found to affect both the internal and external architecture of PMN cells and monocytes in a dose-dependent manner. No effect was observed on lymphocytes. Benzene did not affect the phagocytic activity of PMN cells or monocytes. Preincubation with 2-aminoethylthiosulfuric acid, a free radical scavenger, prevented the observed effects of benzene.

Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain.

Binding and photoaffinity labeling experiments were employed in order to differentiate 1-(1-phenylcyclohexyl)piperidine (PCP) receptor sites in rat brain. Two classes of PCP receptors were characterized and localized: one class binds [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) with high affinity (Kd = 10-15 nM) and the other binds the ligand with a relatively low affinity (Kd = 80-100 nM). The two classes of sites have different patterns of distribution. Forebrain regions are characterized by high-affinity sites (hippocampus greater than frontal cortex greater than thalamus greater than olfactory bulb greater than hypothalamus), but some parts (e.g., hippocampus, hypothalamus) contain low-affinity sites as well. In the cerebellum only low-affinity sites were detected. Binding sites for [3H]PCP and for its photolabile analogue [3H]azido-PCP showed a regional distribution similar to that of the [3H]TCP sites. The neuroleptic drug haloperidol did not block binding to either the high- or the low-affinity [3H]TCP sites, whereas Ca2+ inhibited binding to both. Photoaffinity labeling of the PCP receptors with [3H]AZ-PCP indicated that five specifically labeled polypeptides of these receptors (Mr 90,000, 62,000, 49,000, 40,000, and 33,000) are unevenly distributed in the rat brain. Two of the stereoselectively labeled polypeptides (Mr 90,000 and 33,000) appear to be associated with the high- and low-affinity [3H]TCP-binding sites; the density of the Mr 90,000 polypeptide in various brain regions correlates well with the localization of the high-affinity sites, whereas the density of the Mr 33,000 polypeptide correlates best with the distribution of the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Phencyclidine iminium ion. NADPH-dependent metabolism, covalent binding to macromolecules, and inactivation of cytochrome(s) P-450.

The phencyclidine iminium ion (PCP-Im+), a potentially reactive 2,3,4,5-tetrahydropyridinium species, is formed by the cytochrome(s) P-450-catalyzed alpha-carbon oxidation of phencyclidine (PCP), a commonly abused psychotomimetic agent. Incubation of PCP-Im+ with liver microsomes obtained from phenobarbital-induced rabbits resulted in over 50% loss of microsomal N-demethylase activity and 30% reduction in cytochrome(s) P-450 content. These effects were concentration-dependent, irreversible, and exhibited pseudo-first order kinetics, characteristics of a mechanism-based enzyme inactivation process. Incubation of 3H-PCP-Im+ with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that also was NADPH-dependent. PCP-Im+ was metabolized by liver microsomes in the presence of NADPH and this metabolism was inhibited by SKF 525A and carbon monoxide. HPLC analysis has led to the preliminary characterization of an oxidized metabolite of PCP-Im+ which also is formed from PCP. These results support the proposal that this tetrahydropyridinium metabolite of PCP is biotransformed in a cytochrome(s) P-450-catalyzed reaction to form reactive species capable of covalent interactions with biomacromolecules.

Enzymatic activity in crude oil contaminated rats.

The activity of enzymes found in the plasma, malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), and enzymes from erythrocytes, glucose-6-phosphate dehydrogenase (G-6-PDH) and catalase, was studied in rats contaminated by crude oil. Crude oil (tube fed) contamination caused a significant increase in MDH and LDH activity 96 hr after contamination while a decrease in activity was noted in 6-6-PDH and catalase. An additional contamination (1 week after the first contamination), measured 96 hr after contamination, caused a relative decrease in MDH and LDH activity while there was a contrasting relative increase in G-6-PDH and catalase activity. After a recovery period of 3 weeks the only significant change was an increase in catalase activity.

A national monitoring system for congenital malformations in Israel.

In 1976 the Department of Maternal and Child Health in Israel established, at minimum cost, a national system for reporting of congenital malformations. The system is based on hospital reporting of all live births through a special form attached to the live birth certificate. Compliance of reporting has reached 80 to 90%. Data obtained are tabulated and circulated monthly. It was found that forms that were received later reported a relatively higher percentage of congenital anomalies.

N-allyl analogues of phencyclidine: chemical synthesis and pharmacological properties.

Several N-allyl derivatives of 1-phenylcyclohexylamine (PCA) were prepared, and their pharmacology was briefly characterized. The mono- and diallyl derivatives had phencyclidine-like activities in mice but were less potent behaviorally than phencyclidine (PCP). None were PCP antagonists. In vitro these compounds were competitive inhibitors of butyrylcholinesterase (BChE) and protected against inhibition by DFP. In addition, these agents displaced tritiated N-methyl-4-piperidyl benzilate from mouse-brain homogenates and inhibited the effects of acetylcholine on isolated guinea pig ileum. None of these in vitro effects correlated with their PCP-like behavioral activity in vivo in mice.

Species differences determine azido phencyclidine labeling pattern in desensitized nicotinic acetylcholine receptors.

Acetylcholine receptor enriched membranes from Torpedo ocellata, Torpedo marmorata and Torpedo californica were studied using [3H] azido-phencyclidine (AZ-PCP). [3H]-PCP binding to receptors from all three species revealed marked similarities. Photoaffinity labeling by [3H]-AZ-PCP resulted in the tagging of mainly alpha, beta and delta subunits in all species. When carbamylcholine was added, it enhanced the labeling of beta subunits in T. ocellata, delta in T. marmorata and alpha in T. californica, suggesting species differences in the photolabeling pattern. Multiple homologous binding sites for PCP between the receptor subunits would allow small variations in receptor structure to be manifested in labeling by AZ-PCP, with no differences in binding and functional properties of the receptors.

Cultures of central neurons grown in the presence of phencyclidine.

In order to study the mode of action of phencyclidine at the cellular level differentiating cultures of dissociated nerve cells from brain and spinal cord have been used. Cells were grown in the presence of 50-500 microM of PCP or tritium labelled PCP, for the periods of 1-11 days. PCP in the concentration of 500 microM caused progressive degeneration of nerve cells, already noticeable after 24 h. No significant morphological changes were observed in cells exposed to PCP at concentrations up to 200 microM. In brain cultures exposed to 200 microM PCP, 50% decrease of AChE activity was observed. No decrease in enzyme activity was found in cultures of spinal cord.

Characterization of the interaction of phencyclidine and its derivatives with the ionic channel of the nicotinic receptor.

(3H)-Phencyclidine (PCP) binds specifically to the cholinergic ionophore in synaptic membranes prepared from Torpedo electric organ. Experiments performed by the centrifugation method establish that the binding is saturable, reversible and selective and can be characterized by a single dissociation constant (3.6 +/- 1.8 microM). The maximal binding capacity is 600 +/- 150 pmol/mg of membrane protein. Bound (3H)-PCP can be displaced by unlabelled PCP and a series of its derivatives. The reactivity of PCP derivatives in binding to (3H)-PCP binding sites, as related to structural changes at the phenyl, piperidyl and cyclohexyl moieties, is discussed.

Long scotophase acclimation increases free urinary catecholamine content in the rat.

1. Acclimation of laboratory rats Rattus norvegicus albino to long scotophase 8L:16D and 4L:20D at an ambient temperature (Ta) of 25 degrees C caused an increase in urinary free catecholamine compared to 12L:12D at Ta = 25 degrees C. 2. Transferring the same individuals from 8L:16D to 12L:12D (at Ta = 25 degrees C) caused a decrease in this factor. 3. The results of this study suggest that acclimation to long scotophase increases sympathetic nervous system and chromaffine cell activities.

Metabolism of phencyclidine. The role of iminium ion formation in covalent binding to rabbit microsomal protein.

Incubation of phencyclidine (PCP) with rabbit liver microsomes and Na14CN resulted in the metabolically dependent formation of a 14C-labeled cyano adduct of the drug. After isolation by HPLC, this compound was identified as the alpha-aminonitrile [1-(1-phenylcyclohexyl)-2-cyanopiperidine] derivative of PCP by use of chemical-ionization and gas-chromatographic coupled electron-impact mass spectrometry. Synthetic alpha-aminonitrile exhibited identical chemical properties and comigrated in HPLC and GLC with the metabolism derived cyano adduct. Molecular identification of the adduct formed by cyanide trapping provided evidence for the formation of an iminium ion during PCP metabolism. Quantitative estimation by HPLC demonstrated that the alpha-aminonitrile accounted for over 50% of the PCP metabolized in 30 min by hepatic microsomes in vitro. Metabolism-dependent covalent binding of [3H]PCP to rabbit liver microsomal proteins was inhibited by cyanide ion in a concentration-dependent manner with an IC50 value of 57 microM. The concentrations of cyanide ion used in these experiments did not significantly inhibit the metabolism of PCP. These results support our suggestions that iminium ion formation may represent an important intermediary step in the metabolism of PCP and that such a reactive electrophilic species may be capable of covalent interactions with nucleophilic groupings on microsomal macromolecules.

Inhibition of rat liver monoamine oxidase by alpha-methyl- and N-propargyl-amine derivatives.

The inhibition of rat liver monoamine oxidase by a number of N-propargyl and alpha-methyl amine derivatives has been examined. The results indicate that alpha-methyl-substituted primary and secondary amine derivatives tend to show selectivity as reversible inhibitors towards the A-form of the enzyme. The structural features that result in selectivity in irreversible inhibitors are less easy to define and substitution of an N-propargyl group into a compound that is a selective reversible inhibitor of monoamine oxidase will not necessarily result in retention of that selectivity. Replacement of the acetylenic group in a B-selective irreversible inhibitor by an ethylenic group resulted in a compound that was a reversible inhibitor showing slight selectivity for the A-form of the enzyme.

Interaction of phencyclidine with mouse neuroblastoma cells.

Growth of mouse neuroblastoma (Nb) cell (clone M1) was not affected by phencyclidine (PCP) concentrations of 10(-6)M up to 2 x 10(-4)M, whereas 10(-3)M PCP caused a 100% inhibition of cell growth. Several PCP analogs, including the quaternary PCP methiodide, exerted effects similar to those of PCP. The uptake of [piperidyl-3,4-3H]PCP ([3H]PCP) by the Nb cells was studied using cell monolayers in Petri dishes. Non-specific entry of PCP into the cells was linear with added substrate but specific uptake exhibited saturation kinetics. The concentration for half-maximum specific uptake was 2 x 10-(5)M, and the capacity of the cells at saturation was 2-3 nmoles [3H]PCP/mg protein, at 22 degrees. The uptake rate constant was 0.2 +/- 0.05 x 10(5) (M-1 min-1) and the dissociation constant was 0.25 +/- 0.05 (min-1). Uptake was temperature dependent and was inhibited by 2,4-dinitrophenol (DNP). This may indicate that this binding represents (at least in part) an active uptake process of PCP into the cells.

Receptor binding and antinociceptive properties of phencyclidine opiate-like derivatives.

The relative potencies of a new series of phencyclidine (PCP) analogs for the displacement of [3H] morphine binding from rat brain homogenates are well correlated with the relative antinociceptive potencies in the test of writhing induced by acetic acid (0.6%). One group of compounds exerts a completely naloxone-reversible analgesic effect, while the effects of a second group are partially reversed by naxolone. These findings and the structural differences between the two groups suggest that their analgesic is mediated through different opiate receptors.

On the opioid nature of phencyclidine and its 3-hydroxy derivative.

Phencyclidine (PCP) and its 3-hydroxy derivative (PCP-3-OH) caused a dose-dependent, naloxone reversible inhibition of the response of the guinea pig ileum to electrical stimulation. Unlike PCP, PCP-3-OH exerted an opioid antagonistic effect in the mouse vas deferens bioassay. Whereas both compounds displayed a high affinity in displacing [3H]SKF-10047 binding to rat brain membranes, PCP-3-OH displayed a high affinity to [3H]morphine receptors also. The mediation of alpha- and mu-receptors in the opioid effects of these drugs is discussed.

New analgesic drugs derived from phencyclidine.

Several esters of 1-(1-phenylcyclohexyl)-4-piperidinol (3), 1-(1-phenylcyclohexyl)-4-phenyl-4-piperidinol (10) and its propionate (11), and 1-(1-phenylcyclohexyl)-4-phenylpiperidine (13) were prepared and characterized. The new compounds, which are derived from phencyclidine, exerted analgesic activity in mice. The most potent is 10, which is twice as active as morphine. The antinociceptive activity of 10, 11, and 13 could be well correlated with their potency in the mouse vas deferens bioassay, and both were completely reversed by naloxone.

Selective acetylenic 'suicide' and reversible inhibitors of monoamine oxidase types A and B.

1 A number of aromatic-N-propargyl (acetylenic) compounds and indoleamines were tested for their inhibitory action on monoamine oxidase (MAO) type A and type B using the substrates 5-hydroxytryptamine (5-HT), beta-phenylethylamine (PEA) and dopamine. 2 Structure activity studies with aromatic-N-propragyl (acetylenic) derivatives have shown that MAO inhibitory potency is least dependent on the aromatic portion of the compounds. N-methylated propargyl derivatives are the most active and replacement of the methyl group with a higher alkyl or aromatic group results in significant reduction of activity. The triple bond in the N-propargyl portion is absolutely essential for activity and must be beta-to the nitrogen. It is the acetylenic group that gives these compounds their irreversible MAO inhibitory property. 3 The present study has indicated that since the acetylenic compounds resemble the enzyme substrates the distance between the aromatic ring and the N-propargyl terminal is crucial in designating the type A or type B MAO inhibitory property. For MAO type A inhibition, a distance equivalent to at least three carbon units is required, while for the inhibition of the B type enzyme this distance can be 1 or 2 carbon units. 4 The compounds AGN-1133 and AGN-1135 show most promise in Parkinson's disease or as anti-depressants because of their irreversible selective type B MAO inhibition in vitro and in vivo. 5 A number of indoleamine derivatives were found to be reversible selective type A inhibitors.

The effect of phencyclidine on noradrenaline uptake by bovine chromaffin granules.

The effects of the psychotomimetic drug, phencyclidine, on the reserpine sensitive uptake of (-)noradrenaline, on the reserpine resistant uptake of tryptamine, and on catecholamine release were studied in vitro using bovine chromaffin granules. Phencyclidine inhibited the uptake of (-)noradrenaline and tryptamine in a concentration-dependent manner. It caused 50% inhibition of (-)noradrenaline uptake at 2 X 10(-4)M and of tryptamine uptake at 7 X 10(-4)M. Release of catecholamines was not affected by phencyclidine at 0 degree C and pH 6--8 in concentrations up to 8 X 10(-3)M, whereas at 37 degrees C the drug (4 X 10(-3)M) caused a release that was increased when the pH was raised from 6 to 8. Since the effects of phencyclidine on chromaffin granule uptake and release are observed at high concentrations of the drug only, there is no evidence that these effects are relevant to the in vivo effects of the drug.