Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with β-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.     

Isolation and identification of urinary metabolites of AF-2 (3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide) in rabbits

After oral administration of AF-2 (3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide) to rabbits, the two unique metabolites, M-I and M-II, were isolated from the urine. M-I, yellow needles of mp 117°, was identified as a new type metabolite of nitrofuran derivative, 2-(β-carboxypropionyl)-3-(5-methylthio-2-furyl) acrylamide by its mass, ir and nmr spectrometries. M-II, yellow solid, appears to be cis-trans isomer of M-I considering from its uv and mass spectral data, and the behavior on tlc.     

Cis-trans isomerization of nitrofuran derivatives by xanthine oxidase

Enzymatic cis-trans isomerization of nitrofuran derivatives was 3-(5-Nitro-2-furyl)-2-(2-furyl)-demonstrated with milk xanthine oxidase. acrylamide (AF-2) and 3-(5-nitro-2-furyl)-2-(5-bromo-2-furyl)acrylamide (NFBFA) were mainly converted from the cis to the trans form by this enzyme supplemented with an electron donor. This enzymatic reaction was further characterized with respect to its cofactor requirements. Finally, a new cis-trans isomerization mechanism, which is based on transfer of a single electron by a nitroreductase such as xanthine oxidase to a nitrofuran derivative to give the anion free radical, was proposed.     

Deacylation of carcinogenic 5-nitrofuran derivatives by mammalian tissues

The deacylations of N-[4-(5-nitro-2-furyl)-2-thiazolyl] fonnamide (FANFT), N-[4-(5-nitro-2-furyl)-2-thiazolyl] acetarnide (NFTA) and formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl] hydrazide (FNT) by liver, kidney, small intestines and stomach of mouse, rat, hamster and guinea pig were investigated. FANFT was deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT). FANFT formamidase activity was higher in the liver and small intestines of mouse, hamster and guinea pig, and small intestines and stomach of rat. There was no detectable FANFT formamidase activity in the stomach of the mouse and hamster. Neither NFTA nor FNT was deacylated by the rodent tissue homogenates studied. It is suggested that (1)4 ANFT is a metabolite of FANFT but not NFTA; (2) 2-hydrazino-4-(5-nitro-2-furyl)thiazole (HNFT) may not be a metabolite of FNT; and (3) the induction of tumors by FANFT, NFTA and FNT may not be due to a common carcinogenic metabolite, although these chemicals demonstrate similar organ specificities in some of these rodents.     

The action of AF 2 on cultured hamster and human cells under aerobic and hypoxic conditions

AF 2 (2-(2-furyl)-3-(5-nitro-furyl)acrylamide) was toxic to Chinese hamster V 79 cells and normal human fibroblasts in aerobic media. However, the toxicity of the drug was increased many times by hypoxia. Similarly, the frequency of AF 2-induced azaguanine- and ouabain-resistant mutants of V 79 cells was much higher in hypoxia than under aerobic conditions. Both hamster V 79 cells and human fibroblasts metabolized AF 2 and other nitrofurans rapidly only under hypoxic conditions. Human fibroblasts were more sensitive to AF 2 both under aerobic conditions and in hypoxia than were V 79 cells under similar conditions. The Chinese hamster cells consistently gave survival curves with marked shoulders while human cells did not. Aerobic cultures of fibroblasts derived from xeroderma pigmentosum (XP) patients were markedly sensitive to AF 2 while fibroblasts from two ataxia telangeictasia patients had normal sensitivity. Under hypoxic conditions the sensitivity of both types of cells was increased but the XP line remained 5--10-fold more sensitive than normal or ataxia cells. These results suggest that the DNA lesions produced by AF 2 may be regarded as similar to those produced by ultraviolet light, at least in terms of their repairability in human cells.     

Facile syntheses for (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid through a common synthetic route

The delta 5,9 fatty acids (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid were synthesized for the first time in four steps (9-12% overall yield) starting from commercially available 2-(2-bromoethyl)-1,3-dioxolane. The synthetic approach provided enough material to corroborate the structure and stereochemistry of (5Z,9Z)-5,9-nonadecadienoic acid which was recently identified in the flowers of Malvaviscus arboreus (Malvaceae). The novel phospholipids 1-hexadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn-glycer o-3-phosphocholine and 1-octadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn- glycero-3-phosphocholine were also synthesized from commercially available L-alpha-phosphatidylcholine (egg yolk) and characterized by positive ion electrospray mass spectrometry. These are the first examples of unsymmetrical phospholipids with saturated fatty acids at the sn-1 position and delta 5,9 fatty acids at the sn-2 position.     

Action of 5-(2-thienyl) valeric acid as a biotin antagonist

5-(2-Thienyl)valeric acid (TVA), a biotin analogue which can be easily prepared through chemical process, inhibited the growth of a biotin synthesizing Rhodotorula glutinis. The growth inhibition was reversed by the addition of biotin. Among biotin intermediates, dethiobiotin and 7,8-diaminopelargonic acid reversed the inhibition by TVA, while 7-keto-8-amino-pelargonic acid and pimelic acid did not. From these results, it was concluded that TVA is a biotin antagonist which probably acts as an inhibitor of biotin biosynthesis.     

Hydroxylated jasmonic acid and related compounds from Botryodiplodia theobromae

From the culture filtrate of the fungus Botryodiplodia theobromae five hydroxylated cyclopentane fatty acids of the jasmonic acid type were isolated and identified as (11 S -(-)-hydroxyjasmonic acid; (11R)-(-)-hydroxyjasmonic acid; (-)-12-hydroxyjasmonic acid; (-)-8ξ-hydroxyjasmonic acid; (-)-3-oxo-2-(1ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid; (-)-3-oxo-2(4ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid. In addition, the corresponding hydroxylated iso-jasmonic acid analogues were found as minor constituents. During silica gel chromatography 11,12-didehydrojasmonic acid, 11ξ-acetoxyjasmonic acid, 3-oxo-2-(4ξ-acetoxy-2Z-pentenyl)cyclopent-1-yl-butyric acid 3-oxo-2-(2Z,4-pentadienyl)cyclopent-1-yl-butyric acid were formed as artefacts.     

Mutagenic effect of orally given AF-2 on embryonic cells in pregnant Syrian hamsters

Pregnant hamsters were given various doses of AF-2 by stomach tube; then the cells of their embryos were isolated and cultured in normal medium. Chromosome preparations were made within 24 h after the start of primary culture, and examined for chromosomal aberrations. Marked chromosomal abnormalities were observed in cells of embryos of animals treated with AF-2 at over 20 mg/kg. Samples of surviving cells were also cultured in normal medium for 48 h, and then selected in medium containing 8AG or 6TG. This treatment with AF-2 caused marked dose-dependent induction of 8AG- or 6TG-resistant mutations: mutant colonies were even obtained after a single treatment with 2 mg of AF-2 per kg. These results show that this is a sensitive and useful mammalian system for detecting environmental mutagens.     

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid in seeds of Lonchocarpus sericeus

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid have been isolated from seeds of Lonchocarpus sericeus. The concentration of each compound was ca 0.5 % of the fresh seed weight.     

Mutagenesis in cultured human diploid cells. III. Induction of 8-azaguanine-resistant mutations by furylfuramide.

Trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furylfuramide: FF or AF2) was tested for ability to induce 8-azaguanine (8AG) resistant mutations in cultured human diploid cells. FF had a relatively severe cytotoxic effect on the cells. From the concentration-survival curve, the D0 value for 2-h treatment with FF was estimated to be 11 mug/ml. When cells were treated with FF at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected in medium containing 8AG at 30 mug/ml, the induced mutation frequency increased gradually with increase in concentration of FF. When cells were treated with FF at 10 mug/ml for 2 h, cultured in normal medium for various periods of mutation expression time, and selected with 8AG at 30 mug/ml, the highest induced mutation frequency was obtained with 48 h of mutation expression time. Microscopic examination of the numbers of cells in colonies indicated that the total number of cells increased by half during this mutation expression time of 48 h.     

Mutagenic effect of orally given AF-2 on embryonic cells in pregnant syrian hamsters

Pregnant hamsters were given various doses of AF-2 by stomach tube; then the cells of their embryos were isolated and cultured in normal medium. Chromosome preparations were made within 24 h after the start of primary culture, and examined for chromosomal aberrations. Marked chromosomal abnormalities were observed in cells of embryos of animals treated with AF-2 at over 20 mg/kg. Samples of surviving cells were also cultured in normal medium for 48 h, and then selected in medium containing 8AG or 6TG. This treatment with AF-2 caused marked dose-dependent induction of SAG- or 6TG-resistant mutations: mutant colonies were even obtained after a single treatment with 2 mg of AF-2 per kg. These results show that this is a sensitive and useful mammalian system for detecting environmental mutagens.     

Saccharopine and 2-aminoadipic acid in Reseda odorata

2(S),2′(S)-N⁶-(2′-Glutaryl)lysine (l-saccharopine) and 2(S)-2-aminoadipic acid have been isolated from Reseda odorata. When traditional isolation procedures are used l-pyrosaccharopine (5(S),5′(S)-N-(5′-amino-5′-carboxy-pentyl)-2-pyrrolidone-5-carboxylic acid) is formed from l-saccharopine by lactamisation. The degree of lactamisation during various isolation steps has been studied, The amino acids were identified by IR and PMR spectroscopy and the configurations established by enzymic and polarimetric analyses. The contents of saccharopine and 2-amino-adipic acid have been determined relative to the total nitrogen content at various stages in the growth cycle of R. odorata.     

The synthesis of imidodiphosphoric acid derivatives as potential substrates in the pyrophosphorolysis reaction

The preparation conditions for dichlorophosphinylphosphorimidic trichloride were optimized. It was used in the synthesis of esters of imidodiphosphoric acid. The interaction of the trichloride with amines resulted in the corresponding amidodiphosphates rather than in the expected amides of imidodiphosphoric acid.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 96–102.Original Russian Text Copyright © 2005 by Zakirova, Golubeva, Shipitsin, Aleksandrova.     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

The chemotaxonomic significance of two bioactive caffeic acid esters,nepetoidins A and B,in the Lamiaceae

A survey of leaf surface constituents in the family Lamiaceae using HPLC with diode array detection revealed the presence of two characteristic phenolic compounds in many species. The distribution of these phenolics in the Lamiaceae was found to be of taxonomic significance, as they were present in the great majority of species investigated for the subfamily Nepetoideae, including representatives of the well-known genera of culinary herbs, mint, rosemary, sage, thyme and basil. In contrast, they were absent from species of the other subfamilies of Lamiaceae studied and from the related families Verbenaceae, Scrophulariaceae, Acanthaceae and Buddlejaceae. The compounds were isolated from Plectranthus crassus and identified by NMR spectroscopy as the known caffeic acid esters (Z,E)-[2-(3,5-dihydroxyphenyl)ethenyl] 3-(3,4-dihydroxyphenyl)-2-propenoate and (Z,E)-[2-(3,4-dihydroxyphenyl)ethenyl] 3-(3,4-dihydroxyphenyl)-2-propenoate, for which the trivial names nepetoidins A and B are proposed. The presence of this pair of caffeic acid esters adds another character to the chemical, palynological and embryological features distinguishing the Nepetoideae from the other subfamilies of Lamiaceae and related families, and supports the view that the Nepetoideae are a specialised and monophyletic group within the family. Nepetoidin B was shown to have a greater antioxidant activity than gallic, rosmarinic and caffeic acids, and showed activity as an insect phagostimulant. Both compounds were antifungal.     

Structures of the N-linked carbohydrate of ascorbic acid oxidase from zucchini

N-glycan moiety of ascorbic acid oxidase from zucchini (Cucurbita pepo) has been described to be a core-pentasaccharide with a xylose [D'Andrea et al. (1988) Glycoconjugate J 5:151-7]. Ascorbic acid oxidase is sometimes used to characterize antibodies directed against carbohydrate determinants on plant glycoproteins. To prevent misinterpretations of immunological data, the structure of the N-glycan of ascorbic acid oxidase has been reinvestigated. The oligosaccharides were released by almond N-glycosidase and analysed as their pyridylamino derivatives by 2D-HPLC and exoglycosidase digestions. The main structure resembled the typical complex plant N-glycan consisting of a core-pentasaccharide decorated with xylose and 3-linked fucose. The other abundant species lacked the fucose residue. Small amounts of these glycans carried a GlcNAc residue on the 6-arm. Therefore, ascorbic acid oxidase will not only react with antibodies directed against the xylosylated region but also with those binding to N-glycans with 3-linked fucose.     

Energization of amino acid transport in energy-depleted Ehrlich cells and plasma membrane vesicles

We redirect attention to contributions to the energization of the active transport of amino acids in the Ehrlich cell, beyond the known energization by down-gradient comigration of Na⁺, beyond possible direct energization by coupling to ATP breakdown, and beyond known energization by exchange with prior accumulations of amino acids. We re-emphasize the uphill operation of System L, and by prior depletion of cellular amino acids show that this system must receive energy beyond that made available by their coupled exodus. After this depletion the Na⁺-independent accumulation of the norbornane amino acid, 2-aminobicycloheptane-2-carboxylic acid becomes strongly subject to stimulation by incubation with glucose. Energy transfer between Systems A and L through the mutual substrate action of ordinary amino acids was minimized although not entirely avoided by the use of amino acid analogs specific to each system.When 2,4-dinitrophenol was included in the depleting treatment, and pyruvate, phenazine methosulfate, or glucose used for restoration, recovery of uptake of the norbornane amino acid was independent of external Na⁺ or K⁺ levels. Restoration of the uptake of 2-(methylamino)isobutyric acid was, however, decreased by omission of external K⁺. Contrary to an earlier finding, restoration of uptake of each of these amino acids was associated with distinct and usually correlated rises in cellular ATP levels. ATP addition failed to stimulate exodus of the norbornane amino acid from plasma membrane vesicles, although either NADH or phenazine methosulfate did stimulate exodus. ATP production and use is thus associated with transport energization, although evidence for a direct role failed to appear.     

Novel derivatives of chitosan and their antifungal activities in vitro

Three kinds of Schiff bases of carboxymethyl chitosan (CMCTS) were prepared, and their antifungal activities were assessed according to Jasso de Rodríguez's method. The results indicated that 2-(2-hydroxybenzylideneamino)-6-carboxymethylchitosan (HNCMCTS) and 2-(5-chloro-2-hydroxybenzylideneamino)-6-carboxymethylchitosan (HCCMCTS) had better inhibitory effects than those of chitosan or CMCTS against Fusarium oxysporium f. sp. vasinfectum, Alternaria solani, and Valsa mali.