A novel assay of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity in cultured cells and its use for evaluation of cadmium(II) inhibition of this activity.

8-Oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a product of oxidative modification of dGTP, thatcan be misincorporated into DNA, causing AT-->CG mutations. Cells are protected against 8-oxo-dGTP by 8-oxo-dGTP 5'-pyrophosphohydrolases (8-oxo-dGTP-ases) that convert it to 8-oxo-dGMP. Thus, inhibition of 8-oxo-dGTPases may lead to cancer. To elucidate the involvement of 8-oxo-dGTPases in carcinogenesis, an assay of the 8-oxo-dGTPase activity is required. This paper presents such an assay developed for Chinese hamster ovary (CHO) cells that can be applied to any biological material. It includes: (i) a convenient method for preparing 8-oxo-2'-deoxyguanosine 5'-phosphates; (ii) an HPLC/UV quantification of 8-oxo-dGTP hydrolysis products and (iii) separation of 8-oxo-dGTPase activity from interfering 8-oxo-dGTP phosphatase(s). The 8-oxo-dGTPase activity of CHO cells depends on magnesium, has a pH optimum of 8.5, Km for 8-oxo-dGTP of 9.3 microM, and is inhibited by 8-oxo-dGDP, the product of interfering 8-oxo-dGTP phosphatases. The latter must be removed from the assayed samples by ultrafiltration through 30 kDa cut-off membranes. The method was used to test the inhibition by cadmium ions of the activity of 8-oxo-dGTPase in CHO cells. The cells cultured with 0.3-3 microM cadmium(II) acetate for up to 24 h had their 8-oxo-dGTPase activity suppressed in a Cd(II) concentration-dependent manner, down to 70% of the control value.     

In-vitro biological evaluation of some ONS and NS donor Schiff's bases and their metal complexes

Complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) with the Schiff bases salicylidene-o-aminothiophenol (H2L) and thiophene-o-carboxaldeneaniline (SB) have been synthesized and characterized by elemental analyses, magnetic measurements, thermogravimetric analyses as well as infrared spectra and reflectance spectra. The nature of the bonding has been discussed on the basis of IR spectral data. Magnetic susceptibility measurements and electronic spectral data suggest a six-coordinated octahedral structure for these complexes. The complexes of Mn(II), Co(II), Ni(II), Cu(II) are paramagnetic, while Zn(II) and Cd(II) are diamagnetic in nature. The complexes were tested for their antimicrobial activities against Salmonella typhi, Escherichia coli and Serratia marcescens using the "Disc Diffusion Method". The results are compared with the standard drug (tetracycline) and show moderate activity.     

Antifungal potential of binary and mixed-ligand complexes of N,2'-diphenyl acetohydroxamic acid.

Chelating potential of N,2'-DPAHA with 3d metal ions such as Cu(II), Ni(II), Zn(II), and Cd(II) in the presence of Gly and Phen has been investigated. These experiments were designed to study the role of the stability of mixed-ligand complexes in the modulation of its fungicidal potential. The mixed-ligand complexes were found to be more stable than binary complexes. Enhanced stability of mixed-ligand complexes of Ni(II), Co(II), Zn(II), and Cd(II) is presumably due to pi-bonding effects. In the stabilization of the Cu(II) mixed-ligand complex system, the Jahn-Tellar effect may play a vital role, in addition to pi-bonding effects. Fungicidal activity of N,2'-DPAHA and its binary complexes with Cu(II), Ni(II), and Co(II) was examined against Fusarium oxysporum using the inhibition zone technique. Binary complexes of Zn(II) and Cd(II) with N,2'-DPAHA and mixed-ligand complexes M(II)-Gly or Phen-N,2'-DPAHA, where M(II) = Cu(II), Ni(II), Zn(II), Co(II), and Cd(II) were screened against Alternaria alternata by slide germination technique. All mixed-ligand complexes exhibited fungicidal activity but did not improve significantly compared to binary complexes. Synergistic action of primary and secondary ligands has increased the stability of the mixed-ligand complex compared to the binary complex (1:1) of the secondary ligand (N,2'-DPAHA), and the fungicidal potential of the mixed-ligand complex involving N,2'-DPAHA as secondary ligand was not increased.     

In-vitro biological evaluation of some ONS and NS donor Schiff's bases and their metal complexes

Complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) with the Schiff bases salicylidene-o-aminothiophenol (H₂L) and thiophene-o-carboxaldeneaniline (SB) have been synthesized and characterized by elemental analyses, magnetic measurements, thermogravimetric analyses as well as infrared spectra and reflectance spectra. The nature of the bonding has been discussed on the basis of IR spectral data. Magnetic susceptibility measurements and electronic spectral data suggest a six-coordinated octahedral structure for these complexes. The complexes of Mn(II), Co(II), Ni(II), Cu(II) are paramagnetic, while Zn(II) and Cd(II) are diamagnetic in nature. The complexes were tested for their antimicrobial activities against Salmonella typhi, Escherichia coli and Serratia marcescens using the “Disc Diffusion Method”. The results are compared with the standard drug (tetracycline) and show moderate activity.     

The effect of histidine on the structure and antitumor activity of metal-5-halouracil complexes

The ternary complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) ions with 5-halouracils, viz., 5-fluorouracil (5FU), 5-chlorouracil (5ClU), and 5-bromouracil (5BrU), and the biologically important ligand L-histidine (HISD) have been synthesized and characterized by elemental analysis, conductance measurements, infrared spectra, electronic spectra, and magnetic moment (room temperature) measurements. On the basis of these studies, the structures of the complexes have been proposed. All these ternary complexes were screened for their antitumor activity against Dalton's lymphoma in C3H/He mice. It was found that only Mn(II)-5BrU-HISD, Co(II)-5BrU-HISD, Cu(II)-5ClU-HISD, Cu(II)-5BrU-HISD, Zn(II)-5FU-HISD, and Zn(II)-5BrU-HISD complexes have significant antitumor activity with T/C greater than 125% (where T and C represent mean lifespan of treated mice and control mice respectively). The Mn(II)-5FU-HISD, Co(II)-5FU-HISD, Co(II)-5ClU-HISD, Ni(II)-5ClU-HISD, Ni(II)-5BrU-HISD, and Zn(II)-5ClU-HISD complexes are also effective antitumor agents, with T/C greater than 115%. The complexes that showed effective antitumor action in vivo were also found to inhibit 3H-thymidine incorporation (DNA replication) in Dalton's lymphoma cells in vitro.     

Cobalt, nickel, copper and cadmium coordination polymers containing the bis(1,2,4-triazolyl)methane ligand

Co(II), Ni(II), Cu(II) and Cd(II) coordination polymers containing the flexible ditopic bis(1,2,4-triazol-1-yl)methane ligand (Btm) have been prepared by reaction of equimolar quantities of the corresponding cobalt, nickel, copper and cadmium salts in EtOH solution. Structure solution and refinement of polycrystalline materials were performed by powder diffraction technique (XRPD), using conventional laboratory data. The results show that architecturally different coordination polymers were obtained depending on the counter-ion employed. The XRPD results show also that non-covalent interactions are driving forces for the occurrence of different structures.     

Preparation by direct metal exchange and kinetic study of active site metal substituted class I and class II Clostridium histolyticum collagenases

Active site metal substitutions for both gamma- and zeta-collagenases from Clostridium histolyticum have been made by direct metal exchange. The incubation of Co(II), Cu(II), Ni(II), Cd(II), and Hg(II) with these native collagenases results in changes in activity that parallel those observed for the reconstitution of the respective apoenzymes with these metal ions. For both collagenases, the exchange reactions with Co(II) and Cu(II) are complete within 1 min. However, the changes in activity observed on addition of Ni(II), Cd(II), and Hg(II) to gamma-collagenase and Cd(II) and Hg(II) to zeta-collagenase are time dependent. The kinetic parameters Kcat and KM have been determined for each of the active metallospecies. The substitution of the active-site metal ion in gamma-collagenase results in changes in both kcat and KM, while the effect observed in zeta-collagenase is primarily on KM. This suggests that there are differences in the mechanisms of these two collagenases, at least with respect to the role of the zinc ion in catalysis.     

The metal specificity and selectivity of ZntA from Escherichia coli using the acylphosphate intermediate

ZntA from Escherichia coli is a P-type ATPase that confers resistance to Pb(II), Zn(II), and Cd(II) in vivo. We had previously shown that purified ZntA shows ATP hydrolysis activity with the metal ions Pb(II), Zn(II), and Cd(II). In this study, we utilized the acylphosphate formation activity of ZntA to further investigate the substrate specificity of ZntA. The site of phosphorylation was Asp-436, as expected from sequence alignments. We show that in addition to Pb(II), Zn(II), and Cd(II), ZntA is active with Ni(II), Co(II), and Cu(II), but not with Cu(I) and Ag(I). Thus, ZntA is specific for a broad range of divalent soft metal ions. The activities with Ni(II), Co(II), and Cu(II) are extremely low; the activities with these non-physiological substrates are 10-20-fold lower compared with the values obtained with Pb(II), Zn(II), and Cd(II). Similar results were obtained with DeltaN-ZntA, a ZntA derivative lacking the amino-terminal metal binding domain. By characterizing the acylphosphate formation reaction in ZntA in detail, we show that a step prior to enzyme phosphorylation, most likely the metal ion binding step, is the slow step in the reaction mechanism in ZntA. The low activities with Ni(II), Co(II), and Cu(II) are because of a further decrease in the rate of binding of these metal ions. Thus, metal ion selectivity in ZntA and possibly other P1-type ATPases is based on the charge and the ligand preference of particular metal ions but not on their size.     

Higher activity of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) coincides with lower background levels of 8-oxo-2'-deoxyguanosine in DNA of fetal compared with maternal mouse organs.

Mammalian homologues of Escherichia coli MutT, a protein having 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity, are thought to play the same role in preventing the incorporation of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) into DNA. One could thus expect that higher activity of 8-oxo-dGTPase should correlate with a lower background level of 8-oxo-dG in nuclear DNA. During transplacental carcinogenesis experiments, in control healthy Swiss mice on day 18 of gestation we found consistently lower levels of 8-oxo-dG in DNA in fetal livers and lungs (1.74+/-0.04 SE and 1.49+/-0.08 SE 8-oxo-dG/10(5) dG, respectively; pooled organs of fetuses of 8 dams) as compared with maternal organs (3.05+/-0.20 SE and 3.08+/-0.17 SE 8-oxo-dG/10(5) dG, respectively; n = 8). The 8-oxo-dGTPase activity determination in the same organs revealed that the lower levels of 8-oxo-dG in fetal DNA did, indeed, coincide with higher 8-oxo-dGTPase activity (48.8+/-2.6 SE and 52.5+/-2.5 SE U/mg protein in livers and lungs, respectively); and vice versa, higher 8-oxo-dG levels in DNA of maternal organs were associated with lower levels of 8-oxo-dGTPase activity (24.3+/-1.3 SE and 4.7+/-0.6 SE U/mg protein, as above). Without excluding other reasons for the relatively low 8-oxo-dG background in DNA of fetal tissues (e.g., higher level of antioxidants and antioxidative enzymes; more efficient DNA repair), this inverse relationship may support or at least does not contradict the concept of a guardian role of 8-oxo-dGTPase against 8-oxo-dGTP mutagenicity in mammalian cells.     

Preparation and reconstitution with divalent metal ions of class I and class II Clostridium histolyticum apocollagenases

Both gamma- and zeta-collagenases from Clostridium histolyticum are fully and reversibly inhibited by 1,10-phenanthroline at pH 7.5 in the presence of 10 mM CaCl2 with KI values of 0.11 and 0.040 mM, respectively. The inhibition is caused by removal of the single, active-site Zn(II) present in each of these enzymes. The nonchelating analogue 1,5-phenanthroline has no effect on the activity of either enzyme. Dialysis of the enzymes in the presence of 1,10-phenanthroline, followed by back dialysis against buffer containing no chelating agent, gives the respective apocollagenases. Both apoenzymes can be instantaneously and fully reactivated by the addition of 1 equiv of Zn(II). Variable amounts of activity are restored to both apocollagenases by Co(II) and Ni(II) and to gamma-apocollagenase by Cu(II). The activity titration curve for gamma-apocollagenase with Co(II) and Scatchard plots for the reconstitution of gamma-apocollagenase with Cu(II) and Ni(II) and of zeta-apocollagenase with Ni(II) and Co(II) indicate that all activity changes are the result of binding of a single equivalent of these divalent metal ions at the active site of the collagenases. Cd(II) and Hg(II) do not restore measurable activity to either apoenzyme.     

Activity of the antimutagenic enzyme 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) in cultured chinese hamster ovary cells: effects of cell cycle, proliferation rate, and population density

Mammalian 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolases (8-oxo-dGTPases), such as MTH1, are believed to play the same antimutagenic role as their bacterial homologues, like MutT. Both decompose promutagenic 8-oxo-dGTP, a product of active oxygen's attack on dGTP. It is not known how 8-oxo-dGTPase expression and function are regulated. Therefore, we investigated the effect of cell population density, proliferation rate, and cell cycle phase on 8-oxo-dGTPase specific activity in cultured Chinese hamster ovary K1-BH4 (CHO) cells. With increasing cell population density (from 30 to 95% confluence), the activity of 8-oxo-dGTPase per milligram protein decreased by 33% (p =.007 by ANOVA) while cells shifted by 9% into the G(0)/G(1) phase, with a 5% drop in cells in S phase. Importantly, inhibition of the cells' proliferation rate by calf serum deprivation caused a more dramatic 23% shift toward the G(0)/G(1) phase and a 25% drop in S phase, but had no effect on 8-oxo-dGTPase activity. Likewise, no differences in the enzyme activity were observed within cell populations of different cell cycle phases separated by centrifugal elutriation. Thus, the present results exclude cell cycle-dependent regulation of 8-oxo-dGTPase activity in CHO cells or its simple dependence on proliferation rate. The observed decrease of 8-oxo-dGTPase activity with increasing cell population density might be related to augmentation of cell-to-cell contact.     

The copper-enzyme dopamine beta-monooxygenase: studies on the ability of several metals to inhibit the enzyme activity and to replace the copper

The ability of several metals to inhibit dopamine beta-monooxygenase was measured and compared with their ability to compete with the binding of 64Cu to the water-soluble form of the bovine adrenal enzyme at pH 6.0. In the presence of an optimal concentration of copper (0.5 microM in the present assay system), an inhibition was observed upon addition of Hg(II), Zn(II), or Ni(II). Only a small fraction of the inhibition with these metals may be due to uncoupling of electron transport from hydroxylation. Preincubation of these metals with the Cu-depleted apoenzyme before addition of copper, revealed a stronger inhibition than if copper was added before the other metals. Hg(II), Zn(II), and Ni(II) also compete with the binding of 64Cu(II) to the protein. Hg(II) was the most effective and Ni(II) the least effective of these metals, both with respect to inhibition of the enzyme activity and to prevent the binding of 64Cu(II). Competition experiments on the binding of Zn(II) and 64Cu in the presence and absence of ascorbate, indicated i) a similar affinity of Cu(I) and Cu(II) to the native enzyme, and ii) a more rapid binding of Cu(I) than Cu(II) to the Cu-depleted and Zn-containing enzyme. Al(III), Fe(II), Mg(II), Mn(II), Co(II), Cd(II), and Pb(II) neither inhibited the enzyme activity nor competed with the binding of 64Cu(II) to the protein (Fe(II) was not tested for binding). Of those metals cited above only Cu(II)/Cu(I) was able to reactivate the apoenzyme.     

Synthesis,characterization, biological and catalytic applications of transition metal complexes derived from Schiff base

A novel series of Cu(II), Ni(II), Zn(II), Co(II), and Cd(II) complexes have been synthesized from the Schiff base. Structural features were determined by analytical and spectral techniques like IR, ¹H NMR, UV–vis, elemental analysis, molar electric conductibility, magnetic susceptibility and thermal studies. The complexes are found to be soluble in dimethylformamide and dimethylsulfoxide. Molar conductance values in dimethylformamide indicate the non-electrolytic nature of the complexes. Binding of synthesized complexes with calf thymus DNA (CT DNA) was studied. There is significant binding of DNA in lanes 2, 3, and 5. Lanes 4 and 6 are showing more florescence when compared to the control indicating that these molecules are strongly bound to the DNA by inserting themselves between the two stacked base pairs and exhibiting their original property of fluorescence. Angiogenesis study has revealed that the compounds B-2, B-4 and B-5 have potent antitumor efficacy and activation of antiangiogenesis could be one of the possible underlying mechanisms of tumor inhibition.     

Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model

The influence of long-term exposure to cadmium (Cd) on essential minerals was investigated using a Caco-2 TC7 cells and a multi-analytical tool: microwave digestion and inductively coupled plasma mass spectrometry. Intracellular levels, effects on cadmium accumulation, distribution, and reference concentration ranges of the following elements were determined: Na, Mg, Ca, Cr, Fe, Mn, Co, Ni, Cu, Zn, Mo, and Cd. Results showed that Caco-2 TC7 cells incubated long-term with cadmium concentrations ranging from 0 to 10 μmol Cd/l for 5 weeks exhibited a significant increase in cadmium accumulation. Furthermore, this accumulation was more marked in cells exposed long-term to cadmium compared with controls, and that this exposure resulted in a significant accumulation of copper and zinc but not of the other elements measured. Interactions of Cd with three elements: zinc, copper, and manganese were particularly studied. Exposed to 30 μmol/l of the element, manganese showed the highest inhibition and copper the lowest on cadmium intracellular accumulation but Zn, Cu, and Mn behave differently in terms of their mutual competition with Cd. Indeed, increasing cadmium in the culture medium resulted in a gradual and significant increase in the accumulation of zinc. There was a significant decrease in manganese from 5 μmol Cd/l exposure, and no variation was observed with copper.     

Current aspects in metal genotoxicity

While carcinogenic metal ions are mostly non-mutagenic in bacteria, different types of cellular damage have been observed in mammalian cells, which may account for their carcinogenic potential. Two modes of action seem to be predominant: the induction of oxidative DNA damage, best established for chromium compounds, and the interaction with DNA repair processes, leading to an enhancement of genotoxicity in combination with a variety of DNA damaging agents. In the case of Cd(II), Ni(II), Co(II), Pb(II) and As(III), DNA repair processes are disturbed at low, non-cytotoxic concentrations of the respective metal compounds. Even though different steps in DNA repair are affected by the diverse metals, one common mechanism might be the competition with essential metal ions.     

Antibacterial cobalt (II), copper (II), nickel (II) and zinc (II) complexes of mercaptothiadiazole--derived furanyl, thienyl, pyrrolyl, salicylyl and pyridinyl Schiff bases

A series of Co (II), Cu (II), Ni (II) and Zn (II) complexes of mercaptothiadiazole-derived furanyl, thienyl, pyrrorlyl, salicylyl and pyridinyl Schiff bases were synthesized, characterized and screened for their in vitro antibacterial activity against four Gram-negative, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Shigella fexneri, and two Gram-positive; Bacillus subtilis and Staphylococcus aureous bacterial strains. The results of these studies show the metal complexes to be more antibacterial as compared to the prepared un-complexed Schiff bases.     

Comparison of the toxicity of heavy metals to cabbage growth

Summary Cabbage plants were grown for 55 days with a nutrient solution containing 1 and 10 ppm of V, Cr(III), Cr(VI), Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg(I), orHg(II). A comparison of the plant growth and chemical analysis revealed that Cr(VI), Cu, Cd, and Hg(II) in the solution are most toxic to the plant growth (hence detrimental to the cabbage-head formation) and Mn, Fe, and Zn are less toxic than other heavy metals, and that Mn, Zn, Co, Ni, and Cd and translocated into all the plant organs while V, Cr(III), Cr(VI), Fe, Cu, Hg(I), and Hg(II) are accumulated in the roots.     

DNA damage and 2'-deoxyguanosine oxidation induced by S(IV) autoxidation catalyzed by copper(II) tetraglycine complexes: synergistic effect of a second metal ion

S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).     

Complexes of some trace metal ions with 5-fluorouracil

The preparation of 5-fluorouracil complexes with Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) are reported. The new complexes have been characterised by elemental analysis, solid-state infrared, electronic spectra and magnetic measurements. These measurements suggest that the ligand is bonded to the metal ions through the carbonyl group, behaving as a mono- dentate ligand. On the basis of the ν(OH)bending frequencies and the insolubility of the complexes in common organic solvents, polymeric structures have been proposed for the complexes, with bridging through OH groups. Mn(II), Zn(II) and Cd(II) form four-coordinated complexes, while six coordination numbers have been suggested for Co(II), Ni(II) and Cu(II).