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     

The repair of DNA damage induced in human lymphocytes by gamma rays and fast neurons

When Go human lymphocytes are exposed either to gamma-rays or to d(50)-Be neutrons and then immediately incubated in presence of cytosine arabinoside, the frequency of chromosomal aberrations which is normally observed after radiation exposure only is sharply increased. This enhancement of the aberrations, particularly the dicentrics, is, however, less marked when cytosine arabinoside is administered at longer intervals of time after irradiation. For gamma-rays, the treatment with cytosine arabinoside has no effect on the dicentrics yield when given 5 h after irradiation, indicating that the repair is completed within the 5 h after irradiation and that the lesions are not anymore available to produce exchange aberrations. For d(50)-Be neutrons, the time of repair takes approximately 5 h after a dose of 2.0 Gy, whereas it appears to be shorter (3 h) after a dose of 0.5 Gy.     

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.     

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.     

Nicotinamide stimulates repair of DNA damage in human lymphocytes

Nicotinamide stimulates the amount of DNA repair synthesis that occurs when freshly isolated, normal human lymphocytes are treated with UV irradiation, N-methyl-N′-nitro-N-nitroso guanidine, or dimethyl sulfate. Stimulation of DNA repair synthesis is concentration dependent and reaches a maximum between 2 to 5 mM nicotinamide. In contrast, DNA synthesis in cells that have not been subjected to DNA damage is not affected by nicotinamide at concentrations below 2 mM and is inhibited by concentrations between 2 to 5 mM. In the same concentration range, nicotinic acid has no effect on the rate of DNA synthesis in the presence or absence of DNA damage.     

DNA damage and repair with age in individual human lymphocytes

Previous biochemical studies on DNA repair competence and aging have been limited to techniques, such as alkaline elution or nucleoid sedimentation, involving mass cell populations. These techniques provide no information about the distribution of DNA damage and repair among individual cells and are unlikely to detect age-dependent changes affecting a minor fraction of the cell population. We have recently described a microgel electrophoretic assay (Singh et al., 1988) that measures, at the level of the individual cell, single-strand DNA breaks and alkali-sensitive sites. Here, we employ this method to analyze DNA damage and repair in lymphocytes isolated from the peripheral blood of 31 subjects (23 males and 8 females aged 25-91 years) and exposed in vitro to 200 rads of X-irradiation. While basal (pre-irradiation) levels of damage were independent of the age of the donor, an age-dependent increase in DNA damage was observed immediately following irradiation. For all subjects, the mean level of DNA damage was restored to pre-irradiation control levels within 2 h of incubation at 37 degrees C. However, a distribution analysis of DNA damage among cells within each sample indicated the presence of a few highly damaged cells (4-16%) in the 2-h sample, the occurrence of which was significantly more common among aged individuals. These data indicate an age-related decline in DNA repair competence among a small subpopulation of lymphocytes.     

Fuel smoke condensate induced DNA damage in human lymphocytes and protection by turmeric (Curcuma longa)

Summary Twigs-dry leaves smoke condensate (TDS) was investigated for its DNA damaging activity in human peripheral lymphocytes, by using a sensitive method, fluorescence analysis of DNA unwinding (FADU). An aqueous turmeric component (Aq.T) was studied as a protective agent. TDS at one to 100 folds dilution induced 55% DNA damage at 20 min, while 12-0-tetradecanoylphorbol-13-acetate (TPA) at 10 ng/ml induced only 25% damage. Aq.T at 300 ng/1 afforded 90% protection to DNA against TPA and 65% against TPA. The mechanism of Aq.T protection was investigated by using (i) inhibitors of arachidonate cascade, viz., indomethacin (28 M), NDGA (10 M), DBAP (36 M), (ii) antioxidant enzymes viz., CAT (0.2 U/l), SOD (0.6 U/1), (iii) antioxidants - BHA, curcumin (40 M), mixed gangliosides (20 nM) and protease inhibitor TLCK (100 M). These compounds offered the following extents of protection to DNA against TDS: indomethacin-40%, NDGA-83%, DBAP-70%, SOD-38%, CAT-40%, BHA-38%, curcumin-60%, mixed gangliosides-88%, TLCK-85%. Against TPA as clastogenic agent, the extents of protection were: indomethacin-73%, NDGA-32%, DBAP-72%, SOD-60%, CAT, BHA-negligible, curcumin-23%, mixed gangliosides - 60%, TLCK - 59%. These results indicate that (i) TDS and TPA induce DNA damage possibly by different mechanisms, (ii) Aq.T is a more effective protectant against TDS whereas it is on par with other inhibitors against TPA.Abbreviations FADU Fluoroscence Analysis of DNA Unwinding - Aq.T Aqueous component of turmeric - TDS Twigs-Dry leaves Smoke condensate - PBS Phosphate Buffered Saline, 20 mM, 150 mM NaCl, pH 7.4 - TPA 12-O-Tetradecanoyl Phorbol-13-Acetate - NDGA Nordihydroguaiaretic Acid - DBAP 2,4-Dibromo Acetophenone - CAT Catalase - SOD Superoxide Dismutase - BHA Butylated Hydroxyanisole - TLCK Tosyl Lysyl Chloromethyl Ketone - ROS Reactive Oxygen Species - PAH Polycyclic Aromatic Hydrocarbons - DMSO Dimethyl Sulfoxide - Buffer B 250 mM m-inositol, 10 mM sodium phosphate, 1 mM magnesium chloride, pH 7.3 - BSC Beedi Smoke Condensate - CSC Cigarette Smoke Condensate     

Detection of DNA damage and repair by alkaline elution using human lymphocytes

The repair of DNA alkylation damage in human cells is poorly understood. We have adapted the alkaline elution technique for use with human peripheral blood lymphocytes in culture. We have also established conditions necessary for short-term culture of human lymphocytes. Lymphocyte growth which can be maintained for up to 30 days is dependent upon irradiated TK6 feeder cells and T-cell growth factor (crude TCGF). The amount of damage induced by a given concentration of methyl methane-sulfonate (MMS) is dependent upon cell number per ml of growth medium. The DNA damage measured, in lymphocytes, by alkaline elution is a composite of single strand breaks and alkali-labile lesions. Repair of this damage after appropriate recovery periods is also detectable. The irradiated feeder TK6 cells do not contribute to the number of strand breaks detected or the amount of recovery after treatment. This method offers a quick and reproducible means of detecting DNA damage and repair in human T-lymphocytes.     

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.     

Microcystin-LR induced DNA damage in human peripheral blood lymphocytes

Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10μg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1μg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity.     

Simulated microgravity decreases DNA repair capacity and induces DNA damage in human lymphocytes

The effect of simulated microgravity on DNA damage and apoptosis is still controversial. The objective of this study was to test whether simulated microgravity conditions affect the expression of genes for DNA repair and apoptosis. To achieve this objective, human lymphocyte cells were grown in a NASA‐developed rotating wall vessel (RWV) bioreactor that simulates microgravity. The same cell line was grown in parallel under normal gravitational conditions in culture flasks. The effect of microgravity on the expression of genes was measured by quantitative real‐time PCR while DNA damage was examined by comet assay. The result of this study revealed that exposure to simulated microgravity condition decreases the expression of DNA repair genes. Mismatch repair (MMR) class of DNA repair pathway were more susceptible to microgravity condition‐induced gene expression changes than base excision repair (BER) and nucleotide excision repair (NER) class of DNA repair genes. Downregulation of genes involved in cell proliferation (CyclinD1 and PCNA) and apoptosis (Bax) was also observed. Microgravity‐induced changes in the expression of some of these genes were further verified at the protein level by Western blot analysis. The findings of this study suggest that microgravity may induce alterations in the expression of these DNA repair genes resulting in accumulation of DNA damage. Reduced expression of cell‐cycle genes suggests that microgravity may cause a reduction in cell growth. Downregulation of pro‐apoptotic genes further suggests that extended exposure to microgravity may result in a reduction in the cells' ability to undergo apoptosis. Any resistance to apoptosis seen in cells with damaged DNA may eventually lead to malignant transformation of those cells. J. Cell. Biochem. 107: 723–731, 2009. © 2009 Wiley‐Liss, Inc.     

Influence of polymorphisms in DNA repair genes XPD, XRCC1 and MGMT on DNA damage induced by gamma radiation and its repair in lymphocytes in vitro

DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.     

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.     

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.     

Inhibition of chromosome repair by caffeine or isonicotinic acid hydrazide on chromosome damage induced by mitomycin C in human lymphocytes

The frequency of interchromatic exchanges induced by mitomycin C in cultured human lymphocytes was markedly lowered in the presence of caffeine or isonicotinic acid hydrazide (INH) during a post-treatment period. The autoradiographic experiment showed that the decrease in the exchange frequency did not result from delaying or cell-killing effects by the post-treatment with caffeine or INH. Therefore, it may deduced that the exchange formation closely related to a process sensitive to caffeine or INH.     

Chromosomal damage induced by caprolactam in human lymphocytes

Caprolactam was tested in the in vitro human lymphocyte cytogenetic assay both in the presence and absence of S9 mix at dose levels up to 5500 micrograms/ml using lymphocytes obtained from a male donor and in the presence of S9 mix using lymphocytes obtained from a female donor. Statistically significant increases in chromosomal damage were observed at 5500 micrograms/ml dose level in cells from both donors. This positive response was enhanced by the inclusion of chromosomal gaps in the calculations. It was concluded that caprolactam induces chromosomal damage in human lymphocytes in vitro albeit at comparatively high dose levels.     

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