Lack of induction of somatic aneuploidy in the mouse by nitrilotriacetic acid (NTA)

Nitrilotriacetic acid (NTA) was tested for the induction of aneuploidy in mouse bone marrow cells. Doses of 138 or 275 mg/kg of body weight were intraperitoneally injected 24 h after implantation of a bromodeoxyuridine tablet. Cell-replication kinetics was assessed by comparing the relative percentages of first, second and third metaphases in control and treated samples. The hyperploidy incidence was estimated in second metaphases only, together with the SCE/cell level. Mice injected with 1.8 mg/kg vinblastine (VBL) were used as positive controls. A slight delay of cell cycle was induced by NTA, as shown by regression analysis applied to average generation time values. No increase over the control level was observed for hyperploidy or SCE induction in NTA-treated mice. VBL induced both cell-cycle alteration and a highly significant (P less than 0.001) increase of the hyperploid cell frequency. On the basis of these and previous (Costa et al., 1988) observations it seems that the non-disjunctional activity of NTA in the mouse is confined to meiotic processes.     

Effects of nitrilotriacetic acid on the induction of gene mutations and sister-chromatid exchanges by insoluble chromium compounds

The influence of nitrilotriacetic acid trisodium salt (NTA) on the mutagenic and clastogenic activity of several water-insoluble or poorly soluble chromium compounds was determined by means of the Salmonella/microsome assay (plate test on TA100 strain) and the sister-chromatid exchange (SCE) test in mammalian cell cultures (CHO line). NTA in itself did not induce gene mutations nor did it increase the frequency of SCE. Cr(VI) compounds (Pb, Ba, Zn, Sr and Ca chromates) and an industrial Cr(VI) pigment, chromium orange (containing PbCrO4 PbO), were inactive or scarcely active mutagens in the Salmonella/microsome test when dissolved in water, but they were increasingly mutagenic when solubilized by 0.5 N NaOH or NTA (10 or 100 mg/ml). Also, the mutagenic activity of Cr(VI), contaminating an industrial Cr(III) pigment (chromite), was slightly enhanced by NTA. Mutagenicity of chromates was correlated with the amounts of Cr(VI) solubilized by NTA or alkali, as determined by the colorimetric reaction with diphenylcarbazide and atomic absorption spectrophotometry, and was decreased by incubation with microsomes, due to reduction of Cr(VI) to the genetically inactive Cr(III) form. In the SCE assay, the insoluble or poorly soluble Ba, Zn, Sr and Ca chromates and the insoluble Cr(VI) pigments zinc yellow (containing ZnCrO4 Zn(OH2], chromium yellow and molybdenum orange (both containing PbCrO4) were directly clastogenic due to cellular endocytosis taking place in prolonged treatments, and NTA significantly increased their chromosome-damaging activity.     

Use of nitrilotriacetic acid (NTA) by Pseudomonas species through iron metabolism

Summary Nitrilotriacetic acid (NTA), when added to solid or liquid media, stimulated the growth of Pseudomonas strains, whereas other synthetic iron-chelators, such as ethylenediaminediacetic acid, ethylenediaminetetraacetic acid, ethylenediaminedihydroxyphenyl acetic acid or ethylene glycol-bis-(-aminoethyl ether)-tetraacetic acid, resulted in concentration-dependent growth inhibition. Experimental data such as stimulation of growth in iron-poor media, inhibitory effect on siderophore biosynthesis, promotion of iron-uptake by NTA, together with the inability of the Pseudomonas strains to use NTA as a carbon and/or a nitrogen source, demonstrated that NTA favours the bacterial growth of Pseudomonas through its scavenging properties for iron. Offprint requests to: J.-M. Meyer     

Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications

High-affinity nitrilotriacetic acids (NTA) have great potential in the molecular manipulation of His-tagged proteins. We have developed a facile method to synthesize multivalent NTA and its conjugates. Starting with appropriately protected lysine, we synthesized the mono-NTA synthons functionalized with either an amino group or a carboxylic group. We then obtained tri-NTA through the condensation of the amino NTA and the carboxylic NTA. Using amino tri-NTA as the key intermediate, we synthesized a series of tri-NTA conjugates with a variety of functional units including biotin, dialkyl, fluorescein, and a hydroxybenzimidate moiety. The biotin-tri-NTA was employed to convert a Biacore streptavidin chip into a high-affinity tri-NTA chip. The equilibrium dissociation constants of tri-NTA/His-tagged protein complexes measured by surface plasmon resonance are in the 20 nM range. Histidine(6)-tagged yeast cytosine deaminase (His6-yCD) was incorporated onto the liposome surface by the lipid-tri-NTA conjugate without any activity loss. Fluorescein-tri-NTA formed a stable 1:1 complex with His6-yCD without significant fluorescence quenching. Specific tri-NTA derivatives for the radiolabeling and coupling of two His-tagged proteins to each other are described. Thus, we have added to the toolbox a number of high-affinity tri-NTA adaptors for the manipulation of His-tagged molecules.     

Mutagenicity of lead chromate in Drosophila melanogaster in the presence of nitrilotriacetic acid (NTA)

By using the sex-linked recessive lethal mutation test in Drosophila melanogaster (standard Basc scheme) we analysed the mutagenic effects of treatments by feeding with nitrilotriacetic acid (NTA: 5 X 10(-2) M), with the insoluble Cr(VI) compound lead chromate, PbCrO4 (supernatant of 4.6 X 10(-4)-M suspension in which the actual concentration was 0.06 gamma/ml as Cr(VI)) and with both compounds preincubated at 3 relative ratios (NTA: 5 X 10(-2) M; PbCrO4: 4.6 X 10(-4), 4.6 X 10(-5) and 9.2 X 10(-6) M, respectively). The estimation of mutation frequencies was done at different developmental stages of the germ cells, namely spermatozoa, spermatids and spermatocytes. Ethyl methanesulphonate (EMS: 5 X 10(-3) M) was used as the reference positive control, with clearly mutagenic results. Treatments with NTA or with PbCrO4 alone did not induce any significant increase of the mutation frequency. PbCrO4 at the 3 concentrations tested was completely soluble in the 5 X 10(-2)-M NTA solution, and the mixture of NTA and PbCrO4 induced significant increases of the frequency of sex-linked lethal mutations, with a significant dose-effect relationship with respect to PbCrO4, apparently as a result of the interaction of the compounds and subsequent release of the genotoxic heavy-metal Cr(VI) ions. This result indicates an important synergistic action of NTA with PbCrO4 under the conditions described.     

Microbial degradation of chelating agents used in detergents with special reference to nitrilotriacetic acid (NTA)

The extensive use of phosphate-based detergents and agricultural fertilizers is one of the main causes of the world-wide eutrophication of rivers and lakes. To ameliorate such problems partial or total substitution of phosphates in laundry detergents by synthetic, non-phosphorus containing complexing agents is practiced in several countries. The physiological, biochemical and ecological aspects of the microbial degradation of the complexing agents most frequently used, such as polyphosphates, aminopolycarboxylates (especially of nitrilotriacetic acid), and phosphonates are reviewed.Abbreviations AODC Acridine orange direct counts - ATMP Aminotrimethylphosphonate - DTPA Diethylenetriaminepentaacetate - DTPMP Diethylenetriaminepentamethylphosphonate - EDTA Ethylenediaminetetraacetate - EDTMP Ethylenediaminetetramethylphosphonate - ED3A Ethylenediaminetriacetate - HEDP Hydroxyethylidenediphosphonate - HEDTA Hydroxyethylethylenediaminetriacetate - IDA Iminodiacetate - IFT Immunofluorescence test - MW Molecular weight - NTA Nitrilotriacetate - PA Polyacrylate - PHC Polyhydroxycarboxylate - PMS Phenazine methosulfate - SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis - SPP Tetrasodiumpyrophosphate - STP Pentasodiumtriphosphate     

Immobilization of histidine-tagged proteins by magnetic nanoparticles encapsulated with nitrilotriacetic acid (NTA)-phospholipids micelle

We described the development of functionalized magnetic nanoparticles (MNPs) with PEG-modification, a phospholipids micelle coating, and their use in manipulating histidine-tagged proteins. Highly monodisperse MNPs were synthesized in an organic solvent and could be phase-transferred into an aqueous solution by encapsulating the nanoparticles with a phospholipids micelle. The phospholipids micelle coating rendered the nanoparticles highly water-soluble, and the functional groups of the phospholipids coating allowed for the bioconjugation of various moieties, such as fluorescent molecules and engineered proteins. Functionalized phospholipids, such as nitrilotriacetic acid (NTA)-phospholipids, caused the MNPs to bind and allowed for manipulation of histidine-tagged proteins. Due to their high surface/volume ratio, the MNPs showed better performance (about 100 times higher) in immobilizing engineered proteins than conventional micrometer-sized beads. This demonstrates that MNPs coated with phospholipids micelle can be a versatile platform for the effective manipulation of various kinds of engineered proteins, which is very important in the field of proteomics. It is expected that a combination of MNPs with optical fluorescent molecules can find applications in bimodal (magnetic and optical) molecular imaging nanoprobes.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

The genotoxicity of nitrilotriacetic acid (NTA) in a somatic mutation and recombination test in Drosophila melanogaster

The genotoxicity of a chelating agent, the trisodium salt of nitrilotriacetic acid (NTA), was assessed in a somatic mutation and recombination test (SMART) in Drosophila melanogaster employing the wing hair markers mwh and flr3. The experiments were performed in parallel in two different laboratories (Padua, Italy and Schwerzenbach, Switzerland). The effectively absorbed doses of NTA, which was administered by feeding to larvae, were determined by a sensitive method employing [3H]leucine which allowed individual consumption levels to be measured. The particular pattern of clone induction produced by this compound suggests that NTA is active in inducing mitotic recombination and possibly aneuploidy in somatic cells of Drosophila. This is discussed in relation to the data present in the literature regarding the genotoxicity of NTA in a variety of experimental systems.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Dose-rate effects of gamma-ray-induced mutations in cultured mammalian cells

The dose-rate dependency of three radiobiological parameters, cell killing and mutations resistant to 6-thioguanine (6-TG^r) and to methotrexate (MTX^r), were studied in populations of mouse L5178Y cells exposed to gamma-rays. when the dose rate was reduced from 50 rad/min to 0.8 rad/min, the shape of the dose—response curves changed from sigmoidal to exponential for cell killing, from upward concave to linear in 6-TG^r mutations and remained linear in MTX^r mutations. A linear quadratic model appears capable of explaining the cell killing and 6-TG^r mutations but not the MTX^r mutations.The declining patterns of induced mutation frequencies of 6-TG^r and MTX^r with decreasing dose rate seem to be similar. The addition of DMSO resulted in protection of cells from cell killing, 6-TG^r and MTX^r mutations with acute exposure, but had little effect with chronic exposure. The reduction of mutation frequency of the 6-TG^r marker with chronic exposure was eliminated by holding cells in ice-cold condition during irradiation. These results suggest that there may be two components of induced mutation. One results primarily from repairable damage induced by the indirect action of radiation and shows a clear dose-rate dependency. The other is mainly from non-repairable damage by the direct action of radiation and is only slightly dose rate-dependent. Under chronic exposure conditions, the latter may predominate.     

DNA-fork displacement rates in cultured mammalian cells with mutations in ribonucleotide reductase

DNA-replication fork displacement rates were measured in mouse S49 lymphosarcoma cell lines and in derivatives of those cell lines. One of the derivatives lacks dCMP deaminase activity and two others bear defined mutations in ribonucleotide reductase. We also examined a revertant cell line that was selected from one of the ribonucleotide reductase mutants and has regained normal ribonucleotide reductase activity. Our results show a correlation between decreased fork-displacement rates and alterations in ribonucleotide reductase, suggesting a possible involvement of this enzyme in the replication apparatus.     

Interactions of nitroaromatic compounds with the mammalian selenoprotein thioredoxin reductase and the relation to induction of apoptosis in human cancer cells

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.     

Induction of gene mutations and chromosomal aberrations by simian virus 40 in cultured mammalian cells

• Induction of gene mutations by SV40 was studied in aneuploid human and Chinese hamster cells. In Chinese hamster cells SV40-induced chromosome aberrations were also studied.

• SV40 penetrated into the cells of both lines and induced synthesis of the T antigen. The efficiency of infection in Chinese hamster cells was tested additionallby their ability to form colonies in medium lacking the serum growth factor. The maximal number of cells with growth factor independence was observed on the first day after infection. When hamster cells had been maintained in “factor-free medium” for the first two passages after infection a sub-line was isolated, which synthesized T antigen 60 days after exposure to SV40. This was considered to be an indirect proof of the integration of viral genome into host chromosome.

• A significant increase in the frequency of chromosomal aberrations was detected in SV40-infected Chinese hamster cells. It was observed on the first and second days after treatment. The most numerous were the chromosome and chromatid breaks, which were distributed randomly in 5 morphological groups according to the chromosome length.

• SV40-induced mutations of resistance to 8-AG and 6-MP in human and Chinese hamster cells respectively were detected, when cells were plated in selective medium one to five days after infection. Induction was detected in all the 4 experiments with human cells and in 9 out of 11 experiments with Chinese hamsters cells. Induction was highly significant according to the Wilcoxon test (P>0.99), when the results of all experiments carried out in human and Chinese hamster cells were summarized. Resistance was stable after prolonged cultivation of 13 isolated clones under non-selective conditions.

• It is suggested that viral genome integration, gene mutations and chromosomal aberrations may have common molecular mechanisms. The role of gene mutations in virus-induced carcinogenesis is discussed.

Heat shock induction of intranuclear actin rods in cultured mammalian cells

Incubation of cultured cells of mouse C3H-2K fibroblastic cell line and other mammalian cell lines at 42.0-43.0 degrees C for 30 min or longer caused disintegration of normal actin structures including stress fibers, and induced formation of intranuclear actin paracrystal-like structures, called actin rods. When cells exposed to the elevated temperatures were shifted back to 37 degrees C, normal actin structures were regained. Pretreatment of cells at moderately high temperatures such as 38.5 degrees C inhibited formation of the actin rods upon subsequent exposure to 42.0 degrees C. Neither microtubules nor intermediate filaments were disrupted by the heat treatment. Several heat shock proteins were found to be synthesized under the conditions where actin rods were induced. However, there is no causal relationship between two cellular events, the induction of intranuclear actin rods and the synthesis of heat shock proteins.