Detection of 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP)-DNA adducts in human pancreatic tissues

Recent epidemiological investigations have observed an association between the consumption of grilled or barbecued meat and an increased risk of pancreatic cancer, suggesting that dietary exposure to heterocyclic aromatic amines (HCA) may contribute to the development of this disease. 2-Amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP) is the most abundant HCA found in well-done and grilled meats. To determine whether HCA-induced DNA damage is present in the human pancreas, immunohistochemistry and computer-assisted image analysis were used to measure PhIP-DNA adducts in 54 normal pancreatic tissues (N) from persons without pancreatic cancer and in 38 normal adjacent pancreatic tissues (A) and in 39 cancer tissues (T) from 68 patients with pancreatic adenocarcinoma. PhIP-DNA adducts were detected in 53 N, 34 A and 39 T samples. Mean values (±SD) of the absorbency for PhIP staining were 0.22±0.04, 0.24±0.04, and 0.24±0.03 for N, A, and T samples, respectively (p=0.004). Using the median absorbency (0.21) of the samples from normal controls as the cut-off, 71% of A and 77% of T tissues, compared with 48% of N tissues, were distributed in the higher range (p=0.009). The odds ratio of pancreatic cancer was 3.4 (95% confidence interval 1.5-7.5, p=0.002) for individuals with a higher level of PhIP-DNA adducts. This is the first report of the detection of PhIP-DNA adducts in human pancreatic tissue samples obtained from patients with unknown exposure to HCA. Although limited by the small sample size, these preliminary results suggest that PhIP exposure may contribute to human pancreatic cancer development.     

Metabolic Fluxes Between [14C]2-Deoxy-D-Glucose and [14C]2-Deoxy-D-Glucose-6-Phosphate in Brain In Vivo

The rates of the phosphorylation and dephosphorylation of 2-deoxyglucose were measured in rat brain in vivo using tracer kinetic techniques. The rate constant for each reaction was estimated from two separate experiments with different protocols for tracer administration. Tracer amounts of [1-14C]2-deoxyglucose (1 microCi) were injected through the internal carotid artery (intraarterial experiment), or through the atrium (intravenous experiment). Brains were sampled by freeze-blowing at various times after the injection. In the intraarterial experiment, the rate constant for the forward reaction from 2-deoxyglucose to 2-deoxyglucose phosphate was calculated by dividing the initial rate of 2-deoxyglucose phosphate production by the 2-deoxyglucose content in brain. The rate constant for the reverse reaction from 2-deoxyglucose phosphate to 2-deoxyglucose was calculated from the decay constant of 2-deoxyglucose phosphate. The rate constants estimated were 10.1 +/- 1.4%/min (SD) and 3.00 +/- 0.01%/min (SD), respectively, for the forward and reverse reactions. In the intravenous experiment, rate constants for both reactions were estimated by compartmental analysis. By fitting data to program SAAM-27, the rate constants for the forward and reverse reactions were estimated as 11.4 +/- 0.4%/min (SD) and 5.1 +/- 0.4%/min (SD), respectively. The rate constants determined were compared to those for the reactions between glucose and glucose-6-phosphate, estimated previously from labeled glucoses. It is concluded that the rate of glucose utilization measured by the 2-deoxyglucose method reflects the rate of the hexokinase reaction and not the rate of glucose utilization or brain energy utilization.     

Liver Injury Due to 3-Amino-1-methyl-5H-pyrido [4,3-b] indole (Trp-P-2) and Its Prevention by Miso

Trp-P-2(3-amino-1-methyl-5H-pyrido [4,3-b] indole) ingestion for 42 d by C3H/HeJJcl mice caused elevation of serum alanine transaminase (ALT) activity and several signs of liver injury. These alterations were not observed in mice fed the diet supplemented with 10% miso. This suggests a preventive effect of miso as to Trp-P-2 induced liver injury.     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

[3H]N-[1-(2-Thienyl)cyclohexyl]-3,4-Piperidine ([3H]TCP) Binding in Human Frontal Cortex: Decreases in Alzheimer-Type Dementia

We studied [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine [( 3H]TCP) binding to human frontal cortex obtained at autopsy from 10 histologically normal controls and eight histopathologically verified cases with Alzheimer-type dementia (ATD). Extensively washed membrane preparations were used to minimize the effects of endogenous substances. In ATD frontal cortex, the total concentration (Bmax) of [3H]TCP binding sites was significantly reduced by 40-50%. The apparent dissociation constant (KD) values showed no significant change. The reduction in binding capacity was also apparent in Triton X-100-treated membrane preparations, and there was a linear correlation between the number of [3H]TCP binding sites and that of N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding sites. [3H]TCP binding sites spared in ATD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. These results suggest that the primary change in NMDA receptor-ion channel complex in ATD brains is the reduction of its number, possibly reflecting the loss of neurons bearing these receptor complexes, and that the functional linkage within the receptor complexes spared in ATD brains remains normal.     

Synthesis and in vitro antimicrobial activity of novel N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide derivatives of benzothiazole class

In this study, a series of novel 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole (6a–g) and 1,3,4-oxadiazole (7a–g, 8) were synthesized from N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide derivatives of benzothiazole class. Antimicrobial properties of the title compound derivatives were investigated against one Gram (+) bacteria (Staphylococcus aureus), three Gram (?) bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) and five fungi (Candida albicans, Aspergillus niger, Aspergillus flavus, Monascus purpureus and Penicillium citrinum) using serial plate dilution method. The investigation of antibacterial and antifungal screening data revealed that all the tested compounds showed moderate to good inhibition at 12.5–100?µg/mL in DMSO. It has been observed that triazolo-thiadiazole derivatives are found to be more active than 1,3,4-oxadiazole derivatives against all pathogenic bacterial and fungal strains.     

Dual-acting agents that possess reversing resistance and anticancer activities: Design, synthesis, MES-SA/Dx5 cell assay, and SAR of Benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo[3',4':1,2]pyridin[3,4-b]indol-2-substitutedacetates

Based on the structural analysis of fumitremorgin C (FTC), imidazoline and β-carboline amino acid benzylester, 14 novel 2-substitutedtetracyclic derivatives of tetrahydrocarboline 4a–n were prepared. We demonstrated that the exposure of MES-SA/Dx5 cells to some of 4a–n resulted in significant reduction of resistance of the cells against doxorubicin. This reduced resistance was accompanied by lowering of IC₅₀ value to doxorubicin from 1.55 ± 0.26 μmol/L to 0.33 ± 0.05 μmol/L for 2-(2-butyl)-derivative 4c, to 1.03 ± 0.22 μmol/L for 2-methyl-derivative 4d, to 0.46 ± 0.04 μmol/L for 2-benzyl-derivative 4f, to 0.98 ± 0.25 μmol/L for 2-indole-3-yl-methyl-derivative 4h, to 0.36 ± 0.03 μmol/L for 2-benzyloxycarbonylmethyl-derivative 4i, to 0.77 ± 0.08 μmol/L for 2-benzyloxycarbonylethyl-derivative 4j, and to 0.77 ± 0.08 μmol/L for 2-benzyloxycarbonylamino-n-butyl-derivative 4l. Proliferation assays of 4a–n indicated 4c,f,i,j were able to inhibit the proliferation of doxorubicin resistant MES-SA/Dx5 cells. The SAR analysis revealed that the benzylester form and the tetracyclic structure of 4a–n were critical for both sensitizing doxorubicin and the cellular anti-proliferative effect.     

Reactions of hybrid organotellurium ligands 1-(4-methoxyphenyl telluro)-2-[3-(6-methyl-2-pyridyl)propoxy]ethane (L) and 1-ethylthio-2-[2-thienyltelluro]ethane (L) with mercury (II) bromide: formation of complexes and their decomposition

Two tellurium ligands 1-(4-methoxyphenyltelluro)-2-[3-(6-methyl-2-pyridyl)propoxy]ethane (L¹) and 1-ethylthio-2-[2-thienyltelluro]ethane (L²) have been synthesized by reacting nucleophiles [4-MeO-C₆H₄Te⁻] and [C₄H₃S-2-Te⁻] with 2-[3-(6-methyl-2-pyridyl)propoxy]ethylchloride and chloroethyl ethyl sulfide, respectively. Both the ligands react with HgBr₂ resulting in complexes of stoichiometry [HgBr₂ · L¹/L²] (1/4), which show characteristic NMR (¹H and ¹³C{¹H}). On crystallization of 1 from acetone-hexane (2:1) mixture, the cleavage of L¹ occurs resulting in 4-MeOC₆H₄HgBr (2) and [RTe⁺→HgBr₂]Br⁻ (3) (where R = -CH₂CH₂OCH₂CH₂CH₂-(2-(6-CH₃-C₅H₃N))). The 2 is characterized by X-ray diffraction on its single crystal. It is a linear molecule and is the first such system which is fully characterized structurally. The Hg-C and Hg-Br bond lengths are 2.085(6) and2.4700(7) Å. The distance of four bromine atoms (3.4041(7)-3.546(7) Å) around Hg (cis to C) is greater than the sum of van der Waal’s radii 3.30 Å. This mercury promoted cleavage is observed for an acyclic ligand of RArTe type for the first time and is unique, as there appears to be no strong intramolecular interaction to stabilize the cleavage products. The 4 on crystallization shows the cleavage of organotellurium ligand L² and formation of a unique complex [(EtS(CH₂)₂SEt)HgBr(μ-Br)Hg(Br)(μ-Br)₂Hg(Br)(μ-Br)BrHg(EtS(CH₂)₂SEt)] · 2HgBr₂ (5), which has been characterized by single crystal structure determination and ¹H and ¹³C{¹H} NMR spectra. The elemental tellurium and [C₄H₃SCH₂]₂ are the other products of dissociation as identified by NMR (proton and carbon-13). The cleavage appears to be without any transmetalation and probably first of its kind. The centrosymmetric structure of 5 is unique as it has [HgBr₃]⁻ unit, one Hg in distorted tetrahedral geometry and one in pseudo-trigonal bipyramidal one. The molecule of 5 may also be described as having [(EtSCH₂CH₂SEt)HgBr]⁺ [HgBr₃]⁻ units, which dimerize and co-crystallize with two HgBr₂ moieties. There are very weak Hg?Br interactions between co-crystallized HgBr₂ units and rest of the molecule. [Hg(3)-Br(1)/Hg(3)-Br(4) = 3.148(1)/3.216(1) Å]. The bridging Hg?Br distances, Hg(2)-Br(4)′, Hg(2)′-Br(4) and Hg(1)-Br(2), are from 2.914(1) to 3.008(1) Å.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Density of NMDA-Coupled and Uncoupled 1-[1-(2-[3H]Thienyl)cyclohexyl]piperidine Recognition Sites in the Brain and Spinal Cord: Differential Effects of NMDA Agonists and Antagonists

Abstract: Binding of 1-[1-(2-[³H]thienyl)cyclohexyl]piperidine ([³H]TCP) to mouse brain and spinal cord membranes was studied using compounds selective for the NMDA-coupled 1-(1-phenylcyclohexyl)piperidine (PCP) and/or σ recognition sites. In both tissues, [³H]TCP labeled two populations of binding sites. Density of the low-affinity sites was approximately the same in both tissues, but the population of the high-affinity [³H]TCP sites was three times bigger in the brain than in the spinal cord. Self- and cross-displacement studies showed that the high-affinity [³H]TCP binding sites could be identical with NMDA receptor-coupled PCP sites, whereas the low-affinity [³H]TCP sites may be associated with σ binding sites in both tissues. The NMDA-coupled PCP sites labeled in the presence of 6.25 n M [³H]TCP constituted a much higher percentage of the total binding in the brain (75%) than in the spinal cord (44%). Consistent with this, reintroduction of glycine and glutamate significantly increased, but DA antagonists significantly inhibited [³H]TCP binding in the brain but not in the spinal cord. Together, these data suggest that a large component of [³H]TCP-labeled binding sites in the spinal cord may be associated with σ but not the NMDA receptor-coupled PCP sites.     

Functional Expression of House Fly (Musca domestica) Cytochrome P450 CYP6D1 in Yeast (Saccharomyces cerevisiae)

Cytochrome P450 CYP6D1 from the house fly is important in the detoxication of xenobiotics and in resistance to pyrethroid insecticides. In house fly microsomes CYP6D1 requires cytochrome b₅ for the metabolism of some substrates, such as benzo[a]pyrene, but does not require cytochrome b₅ for the metabolism of other substrates such as methoxyresorufin. To examine the molecular mechanisms involved in its metabolism of pyrethroids and other substrates, a system for the heterologous expression of CYP6D1 in the yeast Saccharomyces cerevisiae was developed. Heterologous CYP6D1 can be inducibly expressed by culture in media with galactose as the sole carbon source, and is successfully inserted into the yeast microsomes. CYP6D1 is enzymatically active, as measured by methoxyresorufin-O-demethylation, indicating that CYP6D1 is able to interact with yeast P450 reductase. However, CYP6D1 expression did not result in measurable benzo[a]pyrene hydroxylation, suggesting that CYP6D1 cannot interact with yeast cytochrome b₅, or that there is insufficient cytochrome b₅ in the yeast microsomes to support this CYP6D1-mediated activity. Some suggestions are made for improving the yeast microsomal oxidoreductase environment in order to optimize CYP6D1 function.     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).     

Incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair of mice

The incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair of newborn mice was investigated in order to contribute to the validation of PhIP in hair as a suitable biomarker for human dietary exposure. Black mice (C57BL/6J; 7-9 days old) were given graded doses of [³H]-PhIP subcutaneously during the start of the hair growth period. The distribution of [³H]-PhIP and incorporation into hair were investigated by tape-section autoradiography. Almost all the radioactivity in hair represented PhIP as shown by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). A dose-response proportionality of incorporation into hair was found when incorporation was determined by liquid scintillation counting. Autoradiography showed that PhIP was rapidly cleared from the skin, but remained for at least 28 days in the part of the hair shafts which was formed during the exposure period. The present results obtained using the mouse as a model, further support the suggestion that PhIP in hair may be a suitable biomarker for human exposure to dietary PhIP.     

The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: Inhibition by phthalate esters in vitro

This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB₁) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB₁ receptor agonist [³H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC₅₀s: nBBP 27.4 μM; DnHP 33.9 μM; DnBP 45.9 μM; DEHP 47.4 μM; DiOP 55.4 μM; DnOP 75.2 μM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [³H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC₅₀ at 150 μM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [³H]CP-55940 binding assay were positively correlated with inhibition of CB₁ receptor agonist-stimulated binding of [³⁵S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [³H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [³H]SR141716A showed that phthalates reduce the B_max of radioligand without changing its K_d. DnBP and nBBP also rapidly enhanced the dissociation of [³H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB₁ receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB₁ receptor-dependent behavioral and physiological outcomes in the whole animal.     

Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

Opposite Age-Dependent Changes of α2A-Adrenoceptors and Nonadrenoceptor [3H]Idazoxan Binding Sites (I2-Imidazoline Sites) in the Human Brain: Strong Correlation of I2 with Monoamine Oxidase-B Sites

Abstract: In the postmortem human brain (27 specimens of frontal cortex, Brodmann area 9), the specific binding of the antagonists [³H]RX 821002 (2-methoxyidazoxan) to α_2A-adrenoceptors and that of [³H]idazoxan to l₂-imidazo-line sites (a nonadrenoceptor mitochondrial site) were determined in parallel to study the effect of aging (range, 4–89 years) on both brain proteins. The density of α_2A-adrenoceptors and age were negatively correlated (r=-0.71; p < 0.001). In contrast, the density of l₂-imidazo-line sites was positively correlated with aging (r= 0.59; p < 0.005). The ratio of receptor densities (α_2A/l₂) also showed a marked negative correlation with age (r=-0.76; p < 0.001). In an age-selected group (range, 10–89 years), the density of monoamine oxidase (MAO)-B sites labeled by [³H]Ro 19–6327 (lazabemide) also showed a positive correlation with age (r= 0.80; p < 0.005). In these subjects, the density of l₂-imidazoline sites correlated well with the density of MAO-B sites (r= 0.70; p < 0.005). The ratio of the density of these sites (MAO-B/l₂) did not correlate with the age of the subject at death (r=-0.15). In the human frontal cortex, idazoxan displayed very low affinity (K_i= 89 μM) against the binding of [³H]Ro 19–6327 to MAO-B, which discounted a direct interaction of [³H]idazoxan with the active center of the enzyme and indicated that the l₂-imidazoline site cannot be identified with MAO-B. However, l₂-imidazoline sites and MAO-B show a clear coexpression not only in the human frontal cortex during the process of aging, but also in various brain regions of the human and rat brains. It is suggested that the l₂-imidazoline site has a specific location on glial (astrocyte) cells.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Hypertriglyceridemia in Rats Induced by Consumption of a Food-derived Carcinogen, 2-Amino-1-methyl-phenylimidazo[4,5b]pyridine (PhIP)

2-Amino-1-methyl-phenylimidazo[4,5b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine produced in cooked meat and fish, is known to be a carcinogen for rats and mice. This study provides the first evidence for hypertriglyceridemia in rats exposed to PhIP, suggesting its potential risk to induce not only carcinogenesis, but also atherosclerosis, and highlighting the potential importance of PhIP for humans.