Oncogenic purine derivatives: evidence for a possible proximate oncogen.

Two additional urinary metabolites of the chemical oncogen 3-hydroxyxanthine are now identified as 8-chloroxanthine and 8-methylmercaptoxanthine. Such products are thought to be derived from a reactive intermediate which can be tentatively considered to be a proximate oncogen. Since each of these 8-substituted xanthines has also been obtained in vitro by reactions of 3-acetoxyxanthine with chloride ion or methionine, their production in vivo can be explained as resulting through the metabolic formation of an activated ester with a reactivity similar to that of the chemical model.

Tetrodotoxin: Occurrence in atelopid frogs of Costa Rica.

The potent neurotoxin tetrodotoxin, which has previously been found in puffer fish of the order Tetraordontiformes, a goby (Gobius criniger), and the California newt (Taricha torosa), has now been identified in the skins of frogs of the genus Atelopus from Costa Rica.

Increased incidence of mammary tumors in rats after direct or transplacental exposure to 3-hydroxyxanthine.

Rats were exposed directly or transplacentally to the carcinogenic purine N-oxide 3-hydroxyxanthine (3-OH-X). Among female noninbred Wistar rats given 3-OH-X sc, beginning at suckling or weaning ages, there was a significantly greater proportion of mammary tumor bearers and a higher mean number of mammary tumors per rat than among controls. This effect was seen even at low doses producing no other neoplasms such as hepatic carcinomas or fibrosarcomas and fibromas at the site of injection. Noninbred Sprague-Dawley rats, treated transplacentally with 3-OH-X by ip injection into pregnant females, also also had a significantly greater incidence of mammary tumors than did controls.

3-Hydroxyxanthine: transplacental effects and ontogeny of related sulfate metabolism in rats and mice.

The ontogeny of sulfate metabolism related to the metabolic activation of the carcinogenic purine N-oxide 3-hydroxyxanthine (3-OH-X) was studied in noninbred Sprague-Dawley rats. Sulfotransferase activity toward 3-OH-X was detectable in most fetal livers near term at about 25% of adult values and increased slowly after birth. This activity was also present in placentas. Compared to 3-OH-X sulfotransferase, sulfotransferase activity toward p-nitrophenol was lower in fetal livers and was not detected in placentas. Sulfohydrolase activity toward 3'-phosphoadenosine-5'-phosphosulfate was higher in fetal and newborn livers and in placentas than in adult liver. In a parallel transplacential carcinogenicity assay, a low but significant percentage of male rats exposed as fetuses to multiple high doses of 3-OH-X developed single liver carcinomas. After the lowest transplacental dose, the incidence of degenerative kidney disease in old male offspring was significantly higher than that in controls. In an assay with mice, (C57BL/6 X BALB/c)F1 mice exposed transplacentally to 3-OH-X experienced significantly greater perinatal morality and fewer lung adenomas among the surviviors at 20 months of age than did the controls.

Conversion of the oncogenic 1-methylguanine 3-oxide to 3-hydroxy-1-methylxanthine.

Deamination of the oncogenic 1-methylguanine 3-oxide occurs to a significant extent in rats to yield 3-hydroxy-1-methylxanthine and its metabolites. When 3-hydroxy-1-methylxanthine is administered, 1-methyl-8-methylthioxanthine can be recovered from urine and released from hepatic protein. No 1-methyl-8-methylthioguanine was detected in urine or bound to protein. There is no evidence of significant activation of 1-methylguanine 3-oxide by sulfotransferase, but deamination to the oncogenic 3-hydroxy-1-methylxanthine suffices to explain its oncogenicity.

Oncogenicity of purine 3-oxide and unsubstituted purine in rats.

Injection s.c. of purine 3-oxide into Wistar rats resulted in the appearance of sarcomas and fibromas at the interscapular site of administration, carcinomas in the liver, and a high incidence of s.c. fibromas in the hip at a distance from the site of injection. A small number of liver tumors but not tumors at the injection site appeared in rats to which the parent compound, purine, was administered. Oxidation of purine 3-oxide by xanthine oxidase was found to occur in two steps to yield the potent oncogen 3-hydroxyxanthine. A similar process may occur in vivo since a protein preparation from rat s.c. tissue has similar oxidizing activity.

Differential expression of two sodium channel subtypes in human brain.

Two partial human brain sodium channel cDNA sequences (designated HBSC I and II) have been cloned and mapped to chromosome 2q23-2q24 by chromosome microdissection-PCR (CMPCR). The distribution of HBSC I and II mRNA in human brain was studied by means of a novel approach based on the ligase detection reaction. These studies demonstrate that HBSC I and II mRNA is heterogeneously distributed in brain, and that the relative ratio of the two forms can vary as much as 7-fold between different regions.

Effects of log P and phenyl ring conformation on the binding of 5-phenylhydantoins to the voltage-dependent sodium channel.

Binding to the neuronal voltage-dependent sodium channel (NVSC) was evaluated for 12 5-phenylhydantoins which systematically varied either log P and/or 5-phenyl ring orientation. The linear correlation of log P with in vitro sodium channel binding activity (log IC50) for hydantoins 1-12 and diphenylhydantoin (DPH) (r2 = 0.638) suggested that simple partitioning into the lipid phase is important but not sufficient to account for the effects of hydantoins on the NVSC. Comparisons among different hydantoins with the same log P but different low-energy phenyl ring orientations revealed that, in addition to log P, the correct 5-phenyl orientation is important for efficient binding.

Anticonvulsant activities of phenyl-substituted bicyclic 2,4-oxazolidinediones and monocyclic models. Comparison with binding to the neuronal voltage-dependent sodium channel.

8,9-Dioxo-6-phenyl-1-aza-7-oxabicyclo[4.2.1]nonane (1) and 9,10-dioxo-7-phenyl-1-aza-8-oxabicyclo[5.2.1]decane (2), examples of anti-Bredt bicyclic 2,4-oxazolidinediones, were investigated as anticonvulsants in mice. Compound 2 was the more potent (anti-MES ED50 = 66 mg/kg), and its in vivo anti-MES effect was consistent with its in vitro potency of binding to the voltage-sensitive sodium channel (IC50 = 160 microM for the inhibition of binding of [3H]BTX-B), suggesting that 2 may be a new class I anticonvulsant. Several partial structures of 2, either monocyclic lactams or monocyclic 2,4-oxazolidinediones, were also evaluated in these assays, but no correlation was observed between sodium channel binding and anti-MES effects. A significant finding was that monocyclic 5-alkyl-5-phenyl-2,4-oxazolidinediones provided relatively potent, nontoxic, broad-spectrum anticonvulsants.

Comparative molecular field analysis of hydantoin binding to the neuronal voltage-dependent sodium channel.

Comparative molecular field analysis (CoMFA), a 3-D QSAR technique, is widely used to correlate biological activity with observed differences in steric and electrostatic fields. In this study, CoMFA was employed to generate a model, based upon 14 structurally diverse 5-phenylhydantoin analogues, to delineate structural and electrostatic features important for enhanced sodium channel binding. Correlation by partial least squares (PLS) analysis of in vitro sodium channel binding activity (expressed as log IC50) and the CoMFA descriptor column generated a final non-cross-validated model with R2 = 0.988 for the training set. The final CoMFA model explained the data better than a simpler correlation with log P (R2 = 0.801) for the same training set. The CoMFA steric and electrostatic maps described two general features that result in enhanced binding to the sodium channel. These include a preferred 5-phenyl ring orientation and a favorable steric effect resulting from the C5-alkyl chain. This model was then utilized to accurately predict literature sodium channel activities for hydantoins 14-20, which were not included in the training set. Finally the hydantoin CoMFA model was used to design the structurally novel alpha-hydroxy-alpha-phenylamide 21. Synthesis and subsequent sodium channel evaluation of compound 21 (predicted IC50 = 9 microM, actual IC50 = 9 microM), a good binder to the sodium channel, established that the intact hydantion ring is not necessary for efficient binding to this site. Thus alpha-hydroxy-alpha-phenylamides may represent a new class of ligands that bind with increased potency to the sodium channel.

Sodium channel binding and anticonvulsant activities for the enantiomers of a bicyclic 2,4-oxazolidinedione and monocyclic models.

Racemic 7-phenyl-9,10-dioxo-1-aza-8-oxabicyclo[5.2.1]decane (1), a bicyclic 2,4-oxazolidinedione that we previously reported was a possible sodium channel anticonvulsant, was resolved into its enantiomeric forms, the absolute configurations were determined, and the stereoisomers were evaluated for relative sodium channel binding and whole animal anticonvulsant activities. Similar studies were carried out with two monocyclic models, 5-ethyl-5-phenyl-2,4-oxazolidinedione (2) and 5-ethyl-3-methyl-5-phenyl-2,4-oxazolidinedione (3). None of these isomers exhibited stereoselective effects in the sodium channel assay, and only modest enantioselectivities were observed for 2 and 3 in the anticonvulsant assays. (R)-(-)-1 was, however, 4 times more toxic than (S)-(+)-1 in the rotorod test, and due to its larger protective index, (S)-(+)-1 exhibited greater therapeutic potential than either (R)-(-)-1 or racemic 1.

Bicyclic hydantoins with a bridgehead nitrogen. Comparison of anticonvulsant activities with binding to the neuronal voltage-dependent sodium channel.

The anticonvulsant activity of diphenylhydantoin (DPH or phenytoin) is consistent with its actions on the neuronal voltage-dependent sodium channel. To further elucidate the binding requirements for this site, we synthesized several hydantoin analogs and evaluated these in in vitro sodium channel-binding and/or in vivo whole animal anticonvulsant assays. 5-Pentyl-5-phenylhydantoin (8), the most potent binder to the sodium channel in this study, had the same affinity as DPH (IC50 = 40 microM), revealing that one phenyl ring is sufficient for good interactions. Since our previous studies with monophenyl-substituted bicyclic 2,4-oxazolidinediones suggested that N3-alkylation and the conformational constraint of a 5-alkyl substituent over one face of the oxazolidinedione ring improved activity, we synthesized two examples of analogous bicyclic hydantoins. However, the bicyclic hydantoins were much less potent binders to the neuronal voltage-dependent sodium channel than their monocyclic counterparts. The binding activity for the more potent bicyclic hydantoin, 1,8-diaza-9,10-dioxo-7-phenylbicyclo[5.2.1]decane (4) (IC50 = 427 microM), was comparable to that of the ring-opened, N3-methylated monocyclic hydantoin model, 5-butyl-3-methyl-5-phenylhydantoin (9) (IC50 = 285 microM), and these were 8-11 times less potent than the monocyclic model 8, which contains a free imide NH. Furthermore, 5-butyl-5-phenylhydantoin (7; IC50 = 103 microM) was less potent than 8, suggesting that increased log P may enhance binding. Thus, unlike 2,4-oxazolidinediones, N3-alkylation of hydantoins dramatically decreases sodium channel-binding activity. Bicyclic hydantoin 4 was nevertheless a good anti-MES anticonvulsant in mice (ED50 = 86 mg/kg), although this activity likely results from mechanisms other than interactions at the neuronal voltage-dependent sodium channel. Compound 4 was also relatively neurotoxic (TD50 = 124 mg/kg). These results suggest that the binding of hydantoins to the sodium channel may be enhanced by (a) a free imide NH group and (b) an increased log P. Furthermore, 2,4-oxazolidinediones and hydantoins must either orient differently in the same binding site or interact with different sites on the neuronal voltage-dependent sodium channel.

Gas chromatographic/mass spectrometric evidence for the identification of 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline as a normal constituent of rat brain. Its quantification and comparison to rat whole brain levels of dopamine.

Gas chromatographic/mass spectrometric data are presented which demonstrate the presence of 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (DHTIQ) as a normal constituent of rat brain. The level of DHTIQ was calculated to be 10.0 +/- 3.0 ng/g wet weight (+/- S.D., N = 9) of brain tissue while the level of dopamine (DA) was measured as 1.22 +/- 0.22 microg/g (N = 14). The ratio of DHTIQ:DA was thus observed to be approximately 1:100. The possible formation of DHTIQ in alcoholism and schizophrenia is discussed.

Isolation of a human-brain sodium-channel gene encoding two isoforms of the subtype III alpha-subunit.

Voltage-gated sodium channels are members of a multigene family of transmembrane proteins that are important determinants of electrical excitability in cell membranes. These proteins are typically composed of a large alpha-subunit and one or two beta-subunits. The primary structure of alpha-subunits is highly conserved among different subtypes and different species. Based on the conserved sequences and application of the rapid amplification of cDNA ends (RACE) reaction, we have isolated three overlapping clones from human brain. These sequences share highest homology (89%) to the rat brain subtype III gene and cover a 4.2-kb expanse of the transcript. The 5'-most clone has a translation start site located in the same region as other mammalian brain sodium channel genes. A 92-nucleotide insert was found in domain I at a location previously demarcated by published splice sites in rat brain sodium channels IIN/IIA and IIIN/IIIA. It is most likely that this transcript represents the two isoforms (neonatal and adult) of the human brain sodium channel gene, SCN3A (GenBank accession numbers AF035685 and AF035686). As is the case for rat brain sodium channels IIN/IIA and IIIN/IIIA, these isoforms are generated through an alternative splicing mechanism. The conservation of the exon structure suggests that alternative RNA splicing is a common feature for sodium channel mRNA processing and may play an important role in modulating the channel function.

Identifying the promoter region of the human brain sodium channel subtype II gene (SCN2A).

Sodium channel genes are highly regulated. To begin analyzing the human brain sodium channel subtype II gene, SCN2A, at the transcriptional level, we mapped multiple transcriptional start sites within a 397 bp stretch of the 5'-UTR and -flanking region. When inserted into a basic luciferase reporter vector, this 397 bp region can promote luciferase expression in transiently transfected neuroblastoma cells, but not in non-neuronal cells. Thus, this study provides the initial description of a functional promoter in a human voltage-gated sodium channel gene.

Altered brain sodium channel transcript levels in human epilepsy.

Normal, and perhaps pathological, characteristics of neuronal excitability are related to the distribution and density of voltage-gated ion channels such as the sodium channel. We studied normal and epileptic human brain using the ligase detection reaction to measure the relative quantities of mRNAs encoding sodium channel subtypes 1 and 2. Normal brains exhibited characteristic 1:2 ratios which varied by brain region, but the ratios were invariate among individuals. These normal values were altered as much as threefold in anatomically corresponding regions of epileptic brain tissues. Changes of this magnitude in such a highly conserved value support a potential role for sodium channels in the pathophysiology of epilepsy.

Batrachotoxinin-A N-methylanthranilate, a new fluorescent ligand for voltage-sensitive sodium channels.

Batrachotoxin and its derivatives have become important tools for the study of membrane excitability by virtue of their effects on voltage-sensitive sodium channels. Recent studies have shown that the 2,4-dimethylpyrrole carboxylate ester normally occurring in the 20-alpha position of batrachotoxin may be replaced by a benzoate moiety without loss of activity. We have now extended this series of active batrachotoxin derivatives and report here the synthesis of batrachotoxinin-A 20-alpha-N-methylanthranilate. The new fluorescent compound is highly toxic (LD50 approx. 15 micrograms/kg, i.p. mouse) and binds to the same receptor site of voltage-sensitive sodium channels as batrachotoxin with an equilibrium dissociation constant Kd = 180 nM (in the presence of scorpion toxin). These fluorescence and binding properties have been exploited to achieve successful visualization of sodium channels at mammalian nodes of Ranvier. Batrachotoxinin-A 20-alpha-N-methylanthranilate should prove to be a useful compound for studies of sodium channel distribution and for investigations of the microenvironment of the batrachotoxin binding site.

Batrachotoxinin-A-ortho-azidobenzoate: a photoaffinity probe of the batrachotoxin binding site of voltage-sensitive sodium channels.

Batrachotoxin (BTX) is one of a group of potent lipid-soluble neurotoxins which binds voltage-sensitive sodium channels. Here we show that [3H]batrachotoxinin-A-ortho-azidobenzoate ([3H]BTX-OAB), a photolabile derivative of BTX, binds covalently upon irradiation to the BTX sodium channel site of rat cerebral cortical synaptoneurosomes. Another ligand specific for the BTX sodium channel receptor, batrachotoxinin-A 20-alpha-benzoate (BTX-B), competitively inhibited the specific binding of [3H]BTX-OAB. The specific binding of [3H]BTX-OAB was increased by the addition of Leiurus quinquestriatus quinquestriatus scorpion venom (ScTx) and inhibited by veratridine, a member of the same class of sodium channel activators. Examination of the [3H]BTX-OAB-labeled components revealed that over 90% of the specifically incorporated [3H]BTX-OAB was recovered in lipid extracts of photolabeled synaptoneurosomes. Addition of tetrodotoxin (TTX) to the binding mixture increased the specific incorporation of [3H]BTX-OAB into protein components as much as 15-fold. Increasing the incubation temperature from 25 degree C to 37 degrees C had a similar but less marked effect. We conclude that the BTX binding site lies at a lipid-protein interface and that treatments which induce conformational changes in the sodium channel protein (i.e. addition of TTX) can result in a reorientation of BTX at its binding site relative to the protein and lipid domains of voltage-sensitive sodium channels.

The effect of toxin from Leiurus quinquestriatus scorpion venom on the polymerization and stability of microtubules.

The venom of the scorpion Leiurus quinquestriatus, well-known for its actions on voltage-sensitive sodium channels, has now been shown to have pronounced effects on the in vitro polymerization and stability of neuronal microtubules purified by temperature-dependent cycles of assembly and disassembly. The crude venom, at concentrations as low as 100 micrograms/ml, alters both the extent of tubulin polymerization, as monitored by turbidity, and the appearance of polymerized material under electron microscopic examination. Structures formed in the presence of the venom retain the temperature sensitivity characteristic of normal microtubules, but respond to calcium ions abnormally with a dispersal of ordered structures, as reflected by both increased light scattering and electron microscopic analysis. Fractionation of the crude venom suggested that the active component was the same as the polypeptide neurotoxin which interacts with voltage-sensitive sodium channels and this identity was subsequently verified. Thus, the effect on microtubules of highly purified L. quinquestriatus sodium channel toxin obtained from an independent source was indistinguishable from that of the crude venom. These results indicate that the sodium channel toxin from L. quinquestriatus is also a potent cytoskeletal agent in vitro. This finding may be related to the growing body of evidence suggesting that the neuronal cytoskeleton plays a functional role in the maintenance of membrane excitability.

Sodium channel binding and anticonvulsant activities of hydantoins containing conformationally constrained 5-phenyl substituents.

As a preliminary investigation of the importance of the aromatic ring orientation in interactions of 5-phenylhydantoins with the anticonvulsant site on the neuronal voltage-sensitive sodium channel, two isomeric hydantoins containing conformationally constrained phenyl rings and their monocyclic analogues were synthesized. One, a spirohydantoin (2) derived from alpha-tetralone, contains the plane of the phenyl ring in an orientation approximately perpendicular to that for the hydantoin ring. The other, a tricyclic hydantoin (4) derived from tetrahydroisoquinoline, contains the plane of the phenyl ring in an orientation roughly coplanar with that for the hydantoin ring. These compounds were evaluated in sodium channel binding and whole animal (mice) anticonvulsant assays. In both assays, 4 was significantly more potent than 2, suggesting that the anticonvulsant receptor site on the voltage-sensitive sodium channel may require a specific aromatic ring orientation.