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.     

Effects of the comutagens, harman and norharman, on the interaction of a tryptophan pyrolysis product, 3-amino-1-methyl-5H-pyrido (4,3-b) indole with DNA.

Harman and norharman, known as comutagens of many chemicals, were tested for their effect on the binding to DNA of 3-amino-1-methyl-5H-pyrido(4,3-b)indole, (Trp-P-2), a potent mutagen found with harman and norharman in the pyrolysate of tryptophan (1). We demonstrated that the alteration of the DNA helix by intercalation of these comutagens to DNA does not affect the affinity of this potent mutagen for DNA. Covalent binding, however, was inhibited by the comutagens.     

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole Induces Apoptosis and Necrosis with Activation of Different Caspases in Rat Splenocytes

A dietary carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) at 20 μM activates caspase-3-like proteases as an apoptotic marker in rat splenocytes. The present study demonstrated 100 μM Trp-P-1 induced necrosis with activation of caspase-3-like proteases. The activation in necrosis and apoptosis resulted from the activation of caspase-9 and caspase-8, respectively. Thus, Trp-P-1 induces apoptosis and necrosis with the activation of different caspases.     

Determination of less polar heterocyclic aromatic amines in standardised beef extracts and cooked meat consumed in Austria by liquid chromatography and fluorescence detection

An analysis method was developed for the determination of trace levels of less polar heterocyclic aromatic amines (HAs) in food samples. The development started from a frequently used sample pre-treatment scheme which was slightly improved to make it applicable with high-performance liquid chromatography (HPLC) with fluorescence detection. The method was applied for the analysis of a standardised beef extract containing 5-15 ng/g of HAs and the results are compared with those of the other participants in the same European project. In addition, the method was used for the analysis of less polar HAs in cooked meat consumed in Austria.     

Activation of a mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. Identification of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole and its reaction with DNA

A potent mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), isolated from a tryptophan pyrolysate, was activated metabolically by rat liver microsomes and bound to DNA. An active metabolite formed by rat liver microsomes was identified as 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2). Synthetic N-OH-Trp-P-2 reacted with DNA efficiently after O-acetylation or to a lesser extent under acidic conditions (pH 5.5), but did not react appreciably under neutral conditions. Acid hydrolysis of DNA modified by O-acetylated N-OH-Trp-P-2 (N-OAc-Trp-P-2) gave 3-(8-guanyl)amino-1-methyl-5H-pyrido[4,3-b]indole (Gua-Trp-P-2), which is the main modified base of DNA formed by Trp-P-2 in the presence of microsomes. The glycoside bond of the modified base was found to be cleaved by heating at 100° for 1 hr at pH 7.0. In this way, the modified base was liberated from DNA modified by N-OAc-Trp-P-2 in good yield. N-OAc-Trp-P-2 bound to guanyl cytidine more effectively than to guanylic acid, suggesting that covalent binding with guanyl moiety of DNA involves intercalation of the ultimate mutagen into a base pair.     

The Glycine Site of the N-Methyl-D-Aspartate Receptor Channel: Differences Between the Binding of HA-966 and of 7-Chlorokynurenic Acid

The mechanisms of action of three different glycine-site antagonists of the N-methyl-D-aspartate (NMDA)-receptor channel were analyzed employing [3H]glycine direct binding assays, as well as functional glycine- and glutamate-induced uncompetitive blocker binding assays. The latter assays measure apparent channel opening. All three antagonists tested, viz., 7-chlorokynurenic acid (7-Cl-KYNA), kynurenic acid (KYNA), and 1-hydroxy-3-aminopyrrolidone-2 (HA-966), inhibited the binding of [3H]glycine to the NMDA receptor in a dose-dependent manner. These antagonists also inhibited the glycine-induced increase in accessibility of the uncompetitive blocker [3H]N-[1-(2-thienyl)cyclohexyl]-piperidine ([3H]TCP) to the channel. 7-Cl-KYNA and KYNA, but not HA-966, completely blocked the glutamate-induced binding of [3H]TCP, in a manner similar to the non-competitive manner in which the selective NMDA antagonist D-(-)-2-amino-5-phosphonovaleric acid (AP-5) inhibited glycine-induced [3H]TCP binding. The inhibitory effects of HA-966 and of AP-5 on glutamate-induced [3H]TCP binding were overcome when glutamate concentrations were increased. Of the three antagonists, 7-Cl-KYNA appears to be the most potent (Ki = 0.4-1.0 microM) and the most selective glycine antagonist. KYNA was found to act at both the glycine (Ki = 40-50 microM) and the glutamate sites. In contrast, HA-966 (Ki = 6-17 microM) appears to act either on a domain distinct from the glutamate and the glycine sites, but tightly associated with the latter, or at the glycine site, but according to a mechanism distinct from that of 7-Cl-KYNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory Effects of Ascorbic Acid on the Binding of [3H]Dopamine Antagonists to Neostriatal Membrane Preparations : Relationship to Lipid Peroxidation

Abstract: Ascorbic acid, sodium ascorbate, and isoascorbic acid (the stereo-isomer of ascorbic acid) inhibited the stereospecific binding of [³H]spiroperidol to neostriatal membrane preparations. Greater inhibitory effects were obtained at intermediate concentrations of the three ascorbic acid analogs (i.e., 0.06 and 0.6 mM) than at higher (6 m M ) or lower (0.006 m M ) concentrations. In parallel experiments, the three ascorbic acid analogs induced lipid peroxidation, which was also greater at the two intermediate than at higher or lower concentrations. Several known inhibitors of lipid peroxidation, including propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, α-naphthol, and cobalt chloride, as well as the iron chelating agents EDTA and DETAPAC (diethylenetriaminepentaacetic acid) were able to counteract the effects of the ascorbic acid analogs on both lipid peroxidation and on [³H]spiroperidol binding. These data strongly suggest that an iron-catalyzed lipid peroxidation is responsible for the observed inhibitory effects on binding. In other experiments, neostriatal membrane preparations that were preincubated with ascorbic acid (0.6 m M ) and subsequently washed still had greatly diminished capacity to bind [³H]spiroperidol, indicating that ascorbic acid need not be physically present during the binding assay in order to affect binding. This experimental procedure also appears to be a way in which [³H]spiroperidol binding sites can be inactivated and washed free of the inactivating agent.     

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]8-Hydroxy-2-(Di-n-Propylamino)Tetralin Binding to Pre- and Postsynaptic 5-Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD approximately 2 nM) followed by the cerebral cortex (KD approximately 6 nM) and the striatum (KD approximately 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.     

产γ_内酰胺水解酶菌株的筛选及发酵条件研究

(-)γ-内酰胺是合成两种抗艾滋病药物(-)carbovir和(-)abacavir的重要原料,具有重要的应用价值,微生物酶法拆分( /-)γ-内酰胺生产(-)γ-内酰胺的方法具有良好的应用前景。以N-乙酰苯丙氨酸为唯一碳源,从土壤中筛选得到了69株具有酰胺水解酶活性的菌株,利用液相色谱分析方法确定了其中20株有较高的酰胺水解酶活性,利用手性色谱分析的方法进一步得到了一株具有较高立体选择性,能拆分( /-)γ-内酰胺而获得(-)γ-内酰胺的菌株L29-9,对该菌株的产酶培养基的碳、氮源及初始pH值等进行了研究。结果表明:当选择碳源柠檬酸2g/L、氮源酵母提取物5g/Lp、H 7.0、培养时间40h时,通过完整细胞转化,在30℃下经过12h的反应,产物(-)γ-内酰胺产率达40%,ee值99.5%。     

Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH: ubiquinone oxidoreductase (complex I)

Conditions for the reversible dissociation of flavin mononucleotide (FMN) from the membrane-bound mitochondrial NADH:ubiquinone oxidoreductase (complex I) are described. The catalytic activities of the enzyme, i.e. rotenone-insensitive NADH:hexaammineruthenium III reductase and rotenone-sensitive NADH:quinone reductase decline when bovine heart submitochondrial particles are incubated with NADH in the presence of rotenone or cyanide at alkaline pH. FMN protects and fully restores the NADH-induced inactivation whereas riboflavin and flavin adenine dinucleotide do not. The data show that the reduction of complex I significantly weakens the binding of FMN to protein thus resulting in its dissociation when the concentration of holoenzyme is comparable with K(d ( approximately 10(-8)M at pH 10.0).     

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.     

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.     

Artifactual High-Affinity and Saturable Binding of [3H]5-Hydroxytryptamine Induced by Radioligand Oxidation

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and identification of a humanTRPV1 activating compound from soy sauce

Transient receptor potential vanilloid 1 (TRPV1) was identified as a receptor of capsaicin, which is a pungent ingredient in hot red peppers. Due to its relevance for nociception, a physiological and pharmacological study of TRPV1 has also been developed. Therefore, it is important to enrich scientific knowledge regarding the TRPV1 activating or inhibiting compounds. In this study, we fractionated soy sauce based on the human TRPV1 (hTRPV1) activity using column chromatography and purified 5-(9H-pyrido[3,4-b]indol-1-yl)-2-furanmethanol (perlolyrine) as an hTRPV1-activating compound. Additionally, perlolyrine activates the human transient receptor potential ankyrin 1 (hTRPA1). The EC₅₀ of hTRPV1 and hTRPA1 were 2.87 and 1.67 μmol L^?1, respectively. HPLC quantification of soy sauces showed that they contain 2.22–12.13 μmol L^?1 of perlolyrine. The sensory evaluation revealed that perlolyrine has taste modification effect. The results of this study, for the first time, suggest that perlolyrine induces the activation of hTRPV1 and hTRPA1.     

(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)-N-[4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of gamma-[3H]aminobutyric acid [( 3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50 = 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5-hydroxytryptamine1A receptors was inhibited by NO 328 at 5-30 microM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCl. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl-dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design,synthesis and evaluation of 3-quinoline carboxylic acids as new inhibitors of protein kinase CK2

Abstract

In this article, the derivatives of 3-quinoline carboxylic acid were studied as inhibitors of protein kinase CK2. Forty-three new compounds were synthesized. Among them 22 compounds inhibiting CK2 with IC₅₀ in the range from 0.65 to 18.2?μM were identified. The most active inhibitors were found among tetrazolo-quinoline-4-carboxylic acid and 2-aminoquinoline-3-carboxylic acid derivatives.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Synthesis and PKCtheta inhibitory activity of a series of 4-(indol-5-ylamino)thieno[2,3-b]pyridine-5-carbonitriles

The thieno[2,3-b]pyridine-5-carbonitrile with a 5-indolylamine at C-4 and a phenyl group at C-2 had a moderate activity against PKCθ. Optimization of the groups at C-4 and C-2 led to analog 29, which has an IC₅₀ value of 7.5 nM for the inhibition of PKCθ.