Anthraflavic acid inhibits the mutagenicity of the food mutagen IQ: mechanism of action

The ability of anthraflavic acid to inhibit the mutagenicity of IQ was investigated using the Ames test and employing hepatic activation systems from Aroclor 1254-pretreated rats. Incorporation of anthraflavic acid into the S9 mix caused a concentration-dependent decrease in the mutagenicity of IQ. A similar effect was seen when microsomes only were employed as activation systems. Cytosol, as we have previously demonstrated, potentiated the microsome-mediated mutagenicity of IQ and this potentiation was also inhibited by anthraflavic acid. In contrast, anthraflavic acid had no effect on the mutagenicity of the direct-acting microsome-generated metabolites of IQ. It is concluded that anthraflavic acid is a potent inhibitor of IQ mutagenicity by virtue of its ability to inhibit both its microsomal and cytosolic activation pathways.     

RK bacterial test for independently measuring chemical toxicity and mutagenicity: Short-term forward selection assay

A short-term bacterial assay system for determining the mutagenic potential of environmental substances was developed and validated. Genotoxic activity was demonstrated for selected substances from 10 categories of chemical agents. The RK test results were obtained with one Escherichia coli assay strain that was transiently exposed to, and then removed from the test substance prior to the selection step for mutant cells. The RK test employs a hitherto unused short-term assay technique for selecting forward mutations in the wild-type selector strain cells. The cells of the selector strain are killed upon shifting to 42°C as a consequence of thermal derepression and subsequent expression of the replication genes from an integrated 10-kilobase fragment of phage λ. Cells that acquire mutations in the responsible killing genes are detected by their colony-forming ability at 42°C. A substance is determined to be genotoxic if it is capable of increasing the forward mutation frequency for appearance of these mutant cells. Toxicity of the agent is independently evaluated by examining its effect on the viability of the selector strain at 30°C, when the viral replication genes remain repressed. The flexible assay protocol enables determination of the effect of pH on mutagenic activity, the requirement for metabolic activation, and assays of nearly insoluble or highly toxic substances.     

QSAR study on narcotic mechanism of action and toxicity: a molecular connectivity approach to Vibrio fischeri toxicity testing

Quantitative structure-activity relationships (QSARs) have been established based on narcotic mechanism of action and toxicity data to Vibrio fischeri using molecular connectivity indices. The results obtained suggest that both, the degree of branching and electronic characteristic of the compounds have dominant role in the exhibition of toxicity. The information obtained in the present study will be useful in designing more potent compounds.     

Flavin-dependent substrate photo-oxidation as a chemical model of dehydrogenase action.

As a model of flavin-dependent biological dehydrogenation, flavin-sensitized photodehydrogenation and photodecarboxylation were studied by variation of substrate, flavin, pH and solvent. Evidence for the following rules is given. (1) When the reactive site of a photosubstrate is an alpha-carbon atom of the type CH-CO2-, decarboxylation is preferred over dehydrogenation, whereas the reverse is true for the neutral CH-CO2H. (2) Consequently these reactions do not exhibit a measurable isotope effect with C2H-CO2-, in contrast with the findings by Penzer, Radda, Taylor & Taylor [(1970) Vitam. Horm. (N.Y.) 28, 441--466], which could not be reproduced. When the substate does not contain a carboxylate group, isotope effects occur, in verification of previous reports, e.g. for benzyl alcohol C6H5-C2H20H. (3) The mechanism of flavin-sensitized substrate photodecarboxylation is assumed to consist in a primary carbanion fixation at the flavin nucleus (position 4a, 5 or 8) with concomitant liberation of CO2. This step is followed by rapid fragmentation of the adduct CH-Fl-red., provided that the substrate contains a functional and electron-donating group X, e.g. X = OH, OCH3 or NH2 (but not NH3+ !) in X CH-CO2-. (4) The minimal requirement for flavin-sensitized C-H dehydrogenation is the presence of a hydroxyl group. For example, methanol as substrate and solvent is dehydrogenated at pH sufficiently alkaline for detection of the presence of the active species CH3O-, whereas at more acidic pH substrate dehydrogenation is competing with flavin autophotolysis, which depends on the substituents in the flavin nucleus.     

噻吩磺隆的毒性及致突变性

选用大鼠、豚鼠及家兔,采用经口及皮肤,粘膜染毒途径,研究其急性毒性。同时有Ames试验,小鼠骨髓嗜多染红细胞微核试验及小鼠睾丸初级精母细胞染色体畸变试验进行致突变性研究,了解噻吩磺隆的毒性及致突变性。大鼠急性经口LD50大于5000mg／kg,经皮LD50大于2000mg／kg。家兔皮肤刺激试验阴性,轻度眼刺激性和弱致敏性。Ames试验,微核试验及小鼠睾丸初级精母细胞染色体畸变试验结果均为阴性。结论 噻吩磺隆属低毒性农药,在本实验条件下无致突变作用。     

Predicting carcinogenicity of petroleum distillation fractions using a modified Salmonella mutagenicity assay

The Ames Salmonella/microsomal activation mutagenesis assay has been modified to improve sensitivity and reproducibility to complex mixtures derived from the refining and processing of petroleum. Oil samples were dissolved in cyclohexane and subsequently extracted with dimethyl sulfoxide to produce aqueous compatible solutions which readily interact with tester bacteria. Also, the liver homogenate (S-9) and NADP cofactor concentrations were increased and hamster rather than rat liver S-9 was used. The initial slope of the dose response curve relating mutagenicity (revertants per plate) to the dose of extract added was used as an index of mutagenic activity, this slope was obtained through a computerized curve fitting procedure. The modified assay was used to rank 18 oil samples for mutagenic activity, this ranking correlates highly (r = 0.92) with potency rankings of the same samples previously determined from dermal carcinogenicity bioassays. Sensitivity and reproducibility of the assay are sufficient to permit routine use for detecting potential carcinogenic activity of individual refinery streams and blends which contain components boiling above 500°F.Abbreviations API American Petroleum Institute - B[a]P benzo[a]pyrene - DMSO dimethyl sulfoxide - NADP nicotinamide adenine dinucleotide phosphate - PAH polycyclic aromatic hydrocarbon - S-9 microsomal fraction from rat liver     

Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimurium. Using the Griess reagent test, nitrite ranging from approximately 1.8 to 5.4 mg/g of snuff was found in the polar fraction of extracts. After acid treatment, nitroso compounds in the amount corresponding to the nitrite concentration were detected. The mutagenic potency of the acid-treated extracts was consistent with the content of nitroso compounds generated. Formation of nitroso compounds and the mutagenic activity under acidic conditions was inhibited by ascorbic acid. The results indicate that a nitrosation process was involved in snuff extracts during acid treatment. Studies related to the source of nitrite in tobacco snuff demonstrated that snuff contained bacteria which were able to reduce nitrate to nitrite and that the amount of nitrite in snuff extracts could be further increased by incubation of the extracts with the bacteria. Since snuff contains a considerable amount of nitrate, it seems that reduction of nitrate in snuff to nitrite by bacteria, and nitrosation of certain constituents in snuff by nitrite under acidic conditions to form mutagenic nitroso compounds are possible mechanisms responsible for the acid-mediated mutagenicity of snuff extracts.     

Hypnotic action of benzodiazepines: a possible mechanism

The objective of this investigation was to determine whether the effects of muscimol on benzodiazepine receptor binding relate to the hypnotic activity of nine benzodiazepines (clonazepam, triazolam, diazepam, flurazepam, nitrazepam, oxazepam, temazepam, clobazam, and chlordiazepoxide) and CL 218,872. There was no correlation between the basal receptor binding affinities of the drugs tested and their hypnotic potencies, whereas the benzodiazepine receptor agonists whose receptor bindings are strongly modulated by muscimol possess potent hypnotic activity. These results indicate that benzodiazepine receptors that couple to GABA receptors are involved in the hypnotic activity of the benzodiazepines.     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of -D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V ₁/K _B , for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of -D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pK _a of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pK _a of His516 in the free reduced enzyme is 6.9.     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of beta-D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V1/K(B), for reactions of these substrates were collected from pH 2.5-8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of beta-D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid. Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pKa of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pKa of His516 in the free reduced enzyme is 6.9.     

The mechanism of oxygen toxicity as a model for interpreting certain biometeorological phenomena

The biological effects of fluctuating electromagnetic and electrostatic fields, as well as aerosol ions, are probably paralleled by the effects of oxygen toxicity in the alteration of electrodynamics, energy transfer and free radical reactions within cells. Symptoms and signs of low-level oxygen toxicity have been correlated with biochemical and anatomical changes in man and animals.Alteration of the pulmonary membrane, red blood cell survival and mitochondrial function in several organs has been found in experiments attempting to validate the efficacy of 100% oxygen at 250 mm Hg as a space cabin atmosphere. The significance of these findings to biometeorological problems is discussed.

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Zusammenfassung Die biologischen Wirkungen der Schwankungen elektromagnetischer und elektrostatischer Felder sowie der Ionen im Aerosol sind wahrscheinlich begleitet von Wirkungen der Sauerstoffgiftigkeit bei der Änderung der Elektrodynamik, des Energietransportes und Reaktionen freier Radikale in den Zellen.Symptome und Zeichen leichter Sauerstoffvergiftung wurden mit biochemischen und anatomischen Veränderungen bei Mensch und Tier korreliert.In Versuchen,in denen die Wirkung von 100% Sauerstoff als Atmosphäre in einer Raumkapsel bei 250 mm Hg untersucht werden sollte,wurden Veränderungen an den Atmungsmembranen, des Überlebens der Erythrozyten und der Mitochondienfunktionen in verschiedenen Organen gefunden. Die Bedeutung dieser Ergebnisse für biometeorologische Probleme wird diskutiert.

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Resume L'effet biologique des variations des champs électromagnétique etélectrostatique ainsi que des ions contenus dans l'aérosole est probablement accompagné de variations de toxicité de l'oxygène.Ces variations s'observent lors de modifications électrodynamiques, du transport d'énergie et des réactions de radicaux libres dans les cellules. On a mis en parallèle des symptomes de faible intoxication par l'oxygène d'une part, des modifications bio-chimiques et anatomiques chez l'homme et les animaux d'autre part. Par des recherches sur l'influence d'une ambiance faite d'oxygène pur à 250 mm Hg de pression, on a trouvé une altération des membranes pulmonaires, de la survie des corpuscules rouges du sang et de la fonction mitochondriale dans plusieurs organes. On discute la signification de ces résultats pour la solution de problèmes biométéorologiques.

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These studies were supported by Contract NASr-115, Directorate of Space Medicine Headquarters, Office of Manned Space Flight, National Aeronautics and Space Administration.     

Human bone cell cultures: a new model for studying the mechanism of action of calcitonin

An investigation on cell cultures obtained from temporal human bone fragments showed that they provide a suitable model for studying the mechanism involved in calcitonin action on bone cells. Furthermore they demonstrated: a transitory increase in 45Ca uptake that returned to control values ten minutes after the hormone was added; a relation between 45Ca uptake and increased cAMP concentrations when these were measured at the same time intervals; a reproduction of the salmon calcitonin (sCT) effect after incubation of the cultures with either db-cAMP or db-cGMP and inhibition of 45Ca uptake and parallel decrease in cAMP levels with propanol. These results suggest that in human bone cell cultures, sCT acts as a temporary promoter of 45Ca uptake, probably by activating an adenylate-cyclase system through a beta-receptor.     

Sodium channel activators: model of binding inside the pore and a possible mechanism of action

Sodium channel activators, batrachotoxin and veratridine, cause sodium channels to activate easier and stay open longer than normal channels. Traditionally, this was explained by an allosteric mechanism. However, increasing evidence suggests that activators can bind inside the pore. Here, we model the open sodium channel with activators and propose a novel mechanism of their action. The activator-bound channel retains a hydrophilic pathway for ions between the ligand and conserved asparagine in segment S6 of repeat II. One end of the activator approaches the selectivity filter, decreasing the channel conductance and selectivity. The opposite end reaches the gate stabilizing it in the open state.     

Use of Drosophila deoxynucleoside kinase to study mechanism of toxicity and mutagenicity of deoxycytidine analogs in Escherichia coli

Most bacteria, including Escherichia coli, lack an enzyme that can phosphorylate deoxycytidine and its analogs. Consequently, most studies of toxicity and mutagenicity of cytosine analogs use ribonucleosides such as 5-azacytidine (AzaC) and zebularine (Zeb) instead of their deoxynucleoside forms, 5-aza-2′-deoxycytidine (AzadC) and 2′-deoxy-zebularine (dZeb). The former analogs are incorporated into both RNA and DNA creating complex physiological responses in cells. To circumvent this problem, we introduced into E. coli the Drosophila deoxynucleoside kinase (Dm-dNK), which has a relaxed substrate specificity, and tested these cells for sensitivity to AzadC and dZeb. We find that Dm-dNK expression increases substantially sensitivity of cells to these analogs and dZeb is very mutagenic in cells expressing the kinase. Furthermore, toxicity of dZeb in these cells requires DNA mismatch correction system suggesting a mechanism for its toxicity and mutagenicity. The fluorescence properties of dZeb were used to quantify the amount of this analog incorporated into cellular DNA of mismatch repair-deficient cells expressing Dm-dNK and the results showed that in a mismatch correction-defective strain a high percentage of DNA bases may be replaced with the analog without long term toxic effects. This study demonstrates that the mechanism by which Zeb and dZeb cause cell death is fundamentally different than the mechanism of toxicity of AzaC and AzadC. It also opens up a new way to study the mechanism of action of deoxycytidine analogs that are used in anticancer chemotherapy.     

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity.

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.