Differences in the consequences of the decay of radioactive64Cu atoms incorporated in cells under either in vitro or in vivo conditions

Summary To study the lethal effect of⁶⁴Cu under in vitro conditions, asynchronous mammalian cells were used. A lethal effect does exist as a consequence of the decay itself of a few⁶⁴Cu atoms incorporated in cellular DNA. This lethal effect is characterized by an exponential survival curve with no shoulder and no tail; it exists even for non-dividing cells. The lethal efficiency per decay is very high. To test the⁶⁴Cu lethal effect under in vivo conditions, experiments were performed with ascitic cells developing in mice. In this case, the lethal effect also exists, but it is not a function of the⁶⁴Cu doses injected in the mice. Faced with this puzzling result, a systemic approach was necessary to understand and counteract ascitic cells developing in mice.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

A non-radioactive method for measuring Cu uptake in HepG2 cells

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 +/- 9.46 pmol microg DNA(-1) (week 1) to 155 +/- 8.63 pmol microg DNA(-1) (week 2) and stabilising at 171 +/- 4.82 pmol microg DNA(-1) (week 3). After three weeks of culture with 2 microM 63Cu the 63Cu/65Cu changed from 2.18 +/- 0.05 to 15.3 +/- 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.     

Biochemical studies of human (A549) and Simian (CV1) cells labeled with64Cu,67Cu,and125IUdR

CV1 and A549 cells were grown in the presence of 64Cu porphyric complex, 64CuCl2, or 67CuCl2. Radioactive copper determinations were performed on whole cells and on isolated cellular DNA. 125IUdR was used to calibrate the particular extraction and purification procedures we developed because of the half-lives of 64Cu and 67Cu. The results obtained have shown that some radioactive copper atoms remained firmly bound to the DNA molecule. Their amount was of the same order when using two different DNA isolation methods for the two cell lines studied. No significant differences were found when 64Cu was used as CuCl2 or as porphyric complex.     

利用彗星试验检测Cu2+对驯化蚯蚓的基因损伤

为研究Cu2+对驯化蚯蚓的损伤影响,将赤子爱胜蚓(Eisenia fetida)在非致死浓度(100mgCu2+·kg-1)下驯化培养2周,以未驯化的蚯蚓为对照,测定Cu2+对驯化及未驯化蚯蚓的急性毒性,并通过彗星试验(cometassay)观察铜胁迫下(400mg·kg-1)驯化后蚯蚓基因损伤的动态变化。结果显示:14d时,Cu2+对驯化蚯蚓和未驯化蚯蚓的半致死浓度(LC50)分别为321.83～542.45和230.83～342.91mg·kg-1,驯化后蚯蚓的存活率得到显著提高。彗星试验结果表示:蚯蚓体腔细胞的尾长、尾部DNA含量以及尾矩呈非正态分布,在11和14d时,驯化后的蚯蚓基因损伤程度明显比未驯化蚯蚓低。彗星试验是检测Cu2+对蚯蚓活体基因损伤的有效手段,蚯蚓体的DNA损伤可以作为指示重金属污染物影响的生物标志物。     

Copper isotopes as possible neoplasia biomarkers in captive wild felids

The longevity of zoo animals is increasing due to continuous improvement in husbandry and veterinary medicine. However, increasing age is correlated to a higher prevalence of neoplasia. Despite tremendous improvement in diagnoses and monitoring capacities, cancers are still a challenge for veterinarians within the global zoo community. The recent use of copper isotopes as biomarkers for neoplasia in both human and veterinary medicine is a promising and cost‐effective diagnostic tool. Two hundred and twenty‐nine serum samples from 10 different species of wild felids under human care were processed through mass spectrometry to determine the ratio of heavy and light copper isotopes (⁶⁵Cu/⁶³Cu). The results of this preliminary study exhibit an important variability between felid species, with a ratio ranging between ?1.71 and 0.63. Additionally, copper isotopes seem to be a promising diagnostic tool in monitoring cancer in wild animals, as in human medicine, where the isotopic ratio decreases significantly with time in the presence of a tumor.     

Cu2+-induced permeability of the Escherichia coli cytoplasmic membrane]

Cu(2+)-induced permeability of cytoplasmic membranes of Escherichia coli for different cations and neutral molecules of saccharose was estimated by studying their effect on cell plasmolysis during uncharged exchange of cytoplasmic K+ ions by periplasmic space cations. The addition of copper resulted in the exchange of K+ ions by periplasmic Na+, Tris+, streptomycin2+, Cu2+, Ca2+, Mg2+, Cd2+, and Mn2+. It is concluded that Cu(2+)-induced conducting pathways in bacterial membranes are hydrophilic channels with a radius of approximately 0.5 nm and a nonselective permeability for different cations.     

The effect of temperature on DNA structural transitions under the action of Cu2+ and Ca2+ ions in aqueous solutions

The work examines the structural transitions of DNA under the action of Cu2+ and Ca2+ ions in aqueous solution at temperatures of 29 and 45 degrees C by ir spectroscopy. Upon binding to the divalent ions studied, DNA transits into the compact state both at 29 and 45 degrees C. In the compact state DNA remains in B-form limits. The compaction process is of high positive cooperativity. As temperature increases the divalent metal ion concentration required to induce DNA compaction decreases in the case of Cu(2+)-induced compaction and increases in the case of Ca(2+)-induced compaction. It is suggested that the mechanism of the temperature effect on DNA compaction in the presence of Cu2+ ions possessing higher affinity for DNA bases differs from that of the temperature influence on Ca(2+)-induced DNA compaction. In the case of copper ions the determining factor is the increase of binding constants of the Cu2+ ions interacting with the denatured parts formed on DNA while in the case of calcium ions it is the decreased screening action of counterions upon the increase of their hydration with temperature. The efficiency of divalent metal ions studied in inducing DNA compaction depends on hydration of counterions. DNA compaction occurs in a narrow interval of Cu2+ concentrations. As the Cu2+ ion concentration increases, DNA compaction is replaced with Cu(2+)-induced DNA aggregation. At elevated temperatures Cu(2+)-induced DNA compaction could acquire a phase transition character.     

Regulation of p53 by metal ions and by antioxidants: dithiocarbamate down-regulates p53 DNA-binding activity by increasing the intracellular level of copper.

Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.     

Lethal modifications of DNA via the transmutation of32P and33P incorporated in the genome of the S13 bacteriophage

Summary When circular single-stranded DNA of phage S13 is labelled with³²P or³³P, the transmutations very efficiently bring about a loss of phage infectiousness (efficiency = 1 for³²P and 0.73 for³³P). For both radionuclides, the lethal efficiencies as well as the lethal events are different. In the case of³²P, the lethal event is the loss of the circular integrity of the DNA molecule, occurring as a consequence of a systematic single strand-break caused by each³²P decay (100%). Conversely, in the case of³³P, the lethal events are either a single strand-break (40%) or a local stereochemical modification (33%). The same primary event, the substitution at each³³P decay of a phosphate by a sulfate molecule, leads to one of these lethal events in relation to the decay site. Moreover, neither the phage adsorption nor its genome injection into bacteria depends on the physical state of the genome, and thus lethality is revealed at only the genetic level.     

Cytotoxic copper(II) salicylaldehyde semicarbazone complexes: mode of action and proteomic analysis

The in vitro cytotoxic studies of a series of salicylaldehyde semicarbazones, HOC?H?CH=N-NHCONR? (H?R?) and their Cu(II) complexes on a number of human tumor cell lines were conducted and it was observed that their cytotoxicities were enhanced following complexation to copper. These copper(II) complexes also demonstrated higher in vitro activities than the reference drug, cisplatin, on the tumor cell lines at micro molar range. Apoptotic assays and cell cycle analysis of the copper complexes, [Cu(HBnz?)Cl] and [Cu(HBu?)Cl] revealed that they mediated cytotoxicity in MOLT-4 cells via apoptosis. Further proteomic investigation of [Cu(HBnz?)Cl] and [Cu(HBu?)Cl] with respect to their protein expression profiles associated with their mode of action was conducted. By comparing the expression levels of 33 identified protein spots amongst the respective compound-treated profiles, we identified similarities in protein expression patterns between the two copper(II) complexes. The possible roles of the identified proteins in the execution of apoptosis by these copper(II) complexes are discussed.     

Fe and Cu stable isotopes in archeological human bones and their relationship to sex

Accurate sex assignment of ancient human remains usually relies on the availability of coxal bones or well-preserved DNA. Iron (Fe) and copper (Cu) stable isotope compositions ((56)Fe/(54)Fe and (65)Cu/(63)Cu, respectively) were recently measured in modern human blood, and an unexpected result was the discovery of a (56)Fe-depletion and a (65)Cu-enrichment in men's blood compared to women's blood. Bones, being pervasively irrigated by blood, are expected to retain the (56)Fe/(54)Fe and (65)Cu/(63)Cu signature of blood, which in turn is useful for determining the sex of ancient bones. Here, we report the (56)Fe/(54)Fe, (65)Cu/(63)Cu, and (66)Zn/(64)Zn ratios from a suite of well-preserved phalanxes (n = 43) belonging to individuals buried in the 17th and 18th centuries at the necropolis of Saint-Laurent de Grenoble, France, and for which the sex was independently estimated from pelvic bone morphology. The metals were purified from the bone matrix by liquid chromatography on ion exchange resin and the isotope compositions were measured by multiple-collector inductively coupled plasma mass spectrometry. The results show that, as expected from literature data on blood, male bone iron is depleted in (56)Fe and enriched in (65)Cu relative to female. No sex difference is found in the (66)Zn/(64)Zn ratios of bone. The concentration and isotopic data show no evidence of soil contamination. Four samples of five (77%) can be assigned their correct sex, a result comparable to sex assignment using Fe and Cu isotopes in blood (81%). Isotopic analysis of metals may therefore represent a valid method of sex assignment applicable to incomplete human remains.     

Molecular nature of the mutations in gene ADE2 in Saccharomyces cerevisiae yeasts. III. Determination of the correlation of the GC AT pairs in genes ADE1 and ADE2

Lethal and mutagenic effects and the nature of mutations induced by decay of 32P incorporated into yeast cell DNA as 32P-deoxyguanosine monophosphate (32PdGMP) and 32P-thymidine monophosphate (32P-TMP), were studied. The lethal efficiency per 32P decay is independent of a labelled nucleotide incorporated into DNA. However, the mutagenic efficiency in ADE1, ADE2 genes per 32P decay is approximately 3 times greater for 32PdGMP than for 32P-TMP. This suggests that ADE1, ADE2 genes contain about 3 times more GC base pairs than AT pairs. Variations in a relative frequencies of GC leads to AT and AT leads to GC transitions were obtained depending upon a nucleotide labelled.     

DNA binding and nucleolytic properties of Cu(ii) complexes of salicylaldehyde semicarbazones

The copper(ii) complexes of two salicylaldehyde semicarbazones, HOC(6)H(4)CH[double bond, length as m-dash]N-NHCONR(2) [H(2)Bnz(2) (R = CH(2)Ph) and H(2)Bu(2) (R = Bu)], were evaluated for their DNA binding and cleavage properties by spectrophotometric DNA titration, ethidium bromide displacement assay and electrophoretic mobility shift assay. Results showed that the Cu(ii) complexes can bind to DNA via a partial intercalation mode with binding constants of 1.1 × 10(4) and 9.5 × 10(3) M(-1) for [Cu(HBnz(2))Cl] and [Cu(HBu(2))Cl], respectively. These complexes also cleave DNA in the presence of ascorbic acid, most likely through hydroxyl radicals that are generated via the reduction of a Cu(ii) to a Cu(i) species. The complexes show similar DNA cleavage activity, which is reflected in the similarity of their frontier molecular orbital energies calculated by density functional theory. These results are discussed in relation to the anticancer properties of the complexes.     

DNA crosslinks,single-strand breaks and effects on bacteriophage T4 survival from tritium decay of [2-3H]adenine, [8-3H]adenine and [8-3H]guanine

Escherichia coli and bacteriophage T4 DNA containing [2-³H]adenine accumulated crosslinks between the complementary strands. For T4 DNA stored in frozen solution there were 0.41 to 0.54 crosslinks formed per tritium decay. The crosslinks were demonstrated both by an increased DNA sedimentation rate in alkaline sucrose gradients and by an increasing amount of DNA that renatured quickly after denaturation by heat or alkali. Single-strand breaks were also formed with an efficiency of 0.08 to 0.50 breaks per tritium decay. DNA containing both [8-³H]adenine and [8-³H]guanine showed no crosslinking but did undergo single-strand breaks at a rate of 0.08 per tritium decay. T4 bacteriophage containing [2-³H]adenine lost plaque-forming ability when stored at 4 °C, with 0.34 lethal hits per tritium decay, whereas the same phage labeled with a mixture of [8-³H]adenine and [8-³H]guanine sustained only 0.12 lethal hits per tritium decay. The loss of plaque-forming ability in the latter case is probably due to a radiation effect from the emitted beta particle; the high lethal efficiency for tritium decay at 2-adenine is probably caused either by crosslinks between complementary strands or from some undetected lesion produced in the DNA.     

Different DNA damaging species as a result of oxidation of n-butyraldehyde and iso-butyraldehyde by Cu(II)

The isomers n- and iso-butyraldehyde (BuA) in combination with Cu(II) induced single and double strand breaks in PM2 DNA, whereas the aldehydes, or Cu(II) alone had only negligible effect. The DNA damage was the result of radical oxidations of the aldehydes under formation of Cu(I). Cu(I) formation was independent of molecular oxygen. Extensive DNA degradation was only observed in the presence of molecular oxygen. Characterization of DNA damage pointed to different ultimate DNA damaging species. While catalase and neocuproine inhibited strand break formation induced by iso-BuA/Cu(II) to a high degree, these inhibitors were less effective in the n-BuA/Cu(II) reaction. On the other hand, sodium azide showed a high strand break inhibition in the n-BuA/Cu(II) reaction, but low inhibition in the iso-BuA/Cu(II) reaction. 2-Deoxyguanosine was hydroxylated in the 8-position by iso-BuA/Cu(II) but little reaction occurred with n-BuA/Cu(II). Chemiluminescence was detected during both BuA/Cu(II) reactions, whereby the intensity of the luminescence signal was 3.5-fold higher for n-BuA/Cu(II) than for iso-BuA/Cu(II). We suppose that the copper(II)-driven oxidation of n- and iso-BuA proceeds via different pathways with different DNA damaging consequences. Whereas the oxidation of iso-BuA mainly results in damage by ^·OH-radicals, the oxidation of n-BuA may lead to a radical reaction chain whereby excited states are involved and the resulting DNA-damaging species are not ^·OH-radicals.     

Steric effect on the nuclease activity of Cu(II) complexes with aminoquinoline derivatives

Three copper(II) complexes of aminoquinoline derivatives, l-glycine-N'-8-quinolylamide (L1), l-alanine-N'-8-quinolylamide (L2), and N-(8-quinolyl) pyridine-2-carboxamide (L3) have been shown to cleave plasmid DNA pBR322 and pUC18 with or without the presence of H(2)O(2)/ascorbate. Crystallographic data reveal that the Cu(II) coordination plane in [Cu(L1)(Ac)(H(2)O)] (1) and [Cu(L2)(Ac)] (2) is nearly co-planar with the quinoline ring. The cleavage activity follows the order of complex 1>complex 2>complex 3, which is in agreement with the reverse order of the steric hindrance of the amino-substituent of the ligands. The presence of the standard radical scavengers does not have a clear effect on the cleavage efficiency of the Cu(II) complexes, suggesting the reactive species leading to DNA damage could be DNA-bound copper-centered radicals rather than the free diffusible ones.     

Enhancement of cytotoxicity of bleomycin by dithiocarbamates: formation of bis(dithiocarbamato) cu(II).

Properties of the reactions of dithiocarbamates and their Cu(II) or Fe(III) complexes with Ehrlich cells were determined and related to their effects on the inhibition of cell proliferation caused by bleomycin and Cu bleomycin. In complete culture medium containing Eagle's minimal essential medium plus Earles salts and 2.5% fetal calf serum, dimethyl- and diethyldithiocarbamates and their copper complexes inhibit cell proliferation and cause cell death. The copper complexes are more effective agents. Ferric tris-diethyldithiocarbamate is also a cytotoxic species. In contrast, when cells are exposed to dimethyldithiocarbamate or its copper complex in Ringer's buffer under metal-restricted condition, washed, and then placed in complete medium, the copper complex is much more active in inhibiting cell growth. The difference is magnified when dihydroxyethyldithiocarbamate and N-methylglucamine dithiocarbamate and their copper complexes are compared in complete media. Incubation of bleomycin or copper bleomycin with Ehrlich cells in Ringer's buffer with or without dimethyldithiocarbamate or bis-dimethyldithiocarbamato Cu(II) leads to no enhancement of cytotoxicity from combinations of agents, except when the two copper complexes are present. Diethyl- or dimethyldithiocarbamate readily extracts copper from Cu(II)bleomycin and iron from Fe(III)bleomycin when ethylacetate is present to remove the tris-dithiocarbamato Fe(III) complex from aqueous solution. When bis-dimethyldithiocarbamato Cu(II) is incubated with Ehrlich cells, copper is released from the complex and bound to high molecular weight and metallothionein fractions. A reductive mode of dissociation of the copper complexes in cells is supported by ESR experiments. Reactions of diethyl- and dimethyldithiocarbamato Cu(II) with thiol compounds demonstrates one possible mechanism of reduction of these complexes.     

Cu isotope ratios are meaningful in ovarian cancer diagnosis

BackgroundOvarian cancer diagnosis is currently based on imaging and circulating CA-125 concentrations with well-known limits to sensitivity and specificity. New biomarkers are required to complement CA-125 testing to increase effectiveness. Increases in sensitivity of isotopic separation via multi collector inductively coupled plasma-mass spectrometry have recently allowed highly accurate measurement of copper (Cu) isotopic variations. Studies in breast cancer patients have revealed changes of serum copper isotopic composition demonstrating the potential for development as a cancer biomarker. Evaluating ⁶⁵Cu/⁶³Cu ratios (δ⁶⁵Cu) in serum samples from cancer patients has revealed a strong correlation with cancer development. In this study blood samples from forty-four ovarian cancer patients, and 13 ovarian biopsies were investigated.ResultsHere we demonstrate that changes in Cu isotopes also occurs in ovarian cancer patients. Copper composition determined by multiple collector inductively coupled plasma mass spectrometry revealed that the copper isotopic ratio δ⁶⁵Cu in the plasma of 44 ovarian cancer patient cohort was significantly lower than in a group of 48 healthy donors, and indicated that serum was enriched for ⁶³Cu. Further analysis revealed that the isotopic composition of tumour biopsies was enriched for ⁶⁵Cu compared with adjacent healthy ovarian tissues.ConclusionsWe propose that these changes are due to increase lactate and Cu transporter activities in the tumour. These observations demonstrate that, combined with existing strategies, δ⁶⁵Cu could be developed for use in ovarian cancer early detection.