Evaluation of the cytotoxic and mutagenic potentiality of technetium-99m in Escherichi coli.

Since technetium-99m (99mTc) was introduced in medical research it has become one of the most employed radionuclides in nuclear medicine. 99mTc is ideal for routine use on the labeling of different radiopharmaceuticals due to its favorable characteristics. However, some biological effects have been described. These effects may be related to internal conversion electron and/or Auger electron emissions from 99mTc decay that present high linear energy transfer and can generate reactive oxygen species (ROS) in the medium. We evaluated in Escherichia coli K12S and Salmonella typhimurium TA102, both proficient in DNA repair, contribution of those decay emissions on the cytotoxicity induced by 99mTc, both either by generating lesions on DNA or by inducing alterations at membrane. We also studied the genotoxic and/or mutagenic potentiality of 99mTc, in Salmonella typhimurium, using the Ames test. The results showed that: i/ 99mTc is cytotoxic to the Escherichia coli K12S strains; ii/ this effect is related to the electrons (Auger and internal conversion) emissions, and iii/ the 99mTc is not mutagenic and/or genotoxic, when measured by Ames test.     

Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli

Stannous ion has been used in different sectors of human interest, such as in food industry and in health sciences. Much is known about stannous chloride (SnCl(2)) toxicity, although, there is no general agreement regarding its genotoxicity. Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts have been used in popular medicine. We evaluated the influence of these crude extracts on the survival of the Escherichia coli wild type (AB 1157) strain submitted to SnCl(2) treatment. Reactive oxygen species (ROS) can be generated by a Fenton like reaction induced by SnCl(2). E. coli culture was treated simultaneously with SnCl(2) and a specific extract. Our results showed a reduction of the SnCl(2) effect on the survival of the cultures in presence of the crude extracts. The extract of M. ilicifolia showed the highest level of protection action against the SnCl(2) effect in comparison with the other extracts. This protector effect could due to the redox properties of these crude extracts. The compounds in the crude extracts could (i) chelate stannous ions, protecting them against the oxidation and avoiding the generation of ROS, (ii) be a scavenger of the ROS generated by the SnCl(2) oxidation and/or (iii) have oxidant compounds that could oxidise the stannous ions, abolishing or reducing the SnCl(2) effect.     

Assessment of the effect of antiseizure drugs on the labeling process of red blood cells and plasma proteins with technetium-99m.

It is estimated that about 2.5 million people only in the United States are affected by epilepsy. Labelled red blood cells (RBC) and plasma proteins (PP) are used for several evaluations in nuclear medicine and drugs affecting those labelings have previously been described. The aim of this study was to evaluate whether the most popular antiseizure drugs interfere with the 99mTc labeling process of RBC and PP. Heparinized blood withdrawn from Wistar rats was incubated with phenobarbital (0.2, 2, 20, 200, 2,000 microg/ml), phenytoin (0.15, 1.5, 15, 150, 1,500 microg/ml), carbamazepine (0.7, 7, 70 microg/ml), clonazepam (0.5, 5, 50, 500 microg/ml) or valproic acid (0.5, 5, 50, 500 microg/ml) for I hr. Stannous chloride (SnCl2), in two different concentrations (0.012 or 1.2 microg/ml) and 99mTc were added. Plasma and cellular fractions were isolated by centrifugation, soluble and insoluble fractions were separated by trichloroacetic acid precipitation. The percentage of radioactivity was calculated for each fraction. Statistical analysis was performed with ANOVA and Dunnet tests. The analysis of the results has shown that phenobarbital (2,000 microg/ml) and clonazepam (50 microg/ml) significantly have reduced the RBC labeling efficiency when it was used the optimal SnCl2 concentration (1.2 microg/ml) and clonazepam (5, 50 microg/ml) has significantly decreased the PP labeling efficiency with 99mTc. Phenytoin (1,500 microg/ml) has decreased the RBC labeling efficiency when the experiments were carried out with a small SnCl2 concentration (0.012 microg/ml). We can suggest that with this in vitro assay, at the therapeutic level of phenytoin, phenobarbital, carbamazepine and valproic acid will not interfere on the 99mTc labeling process of RBC. Interference is displayed at higher phenobarbital concentrations (2,000 microg/ml). However, humans do not tolerate this concentration. On the other hand, a decreased RBC and PP labeling efficiency with 99mTc may be expected for clonazepam at therapeutic levels.     

Spin-trapping studies with MTDO, a dihydroquinoline type radical scavenger

The effect of MTDQ, a dihydroquinoline type free radical scavenger was studied in biological membranes, and model systems. Using spin-labelling and spin-trapping methods, it was that MTDQ induced a decrease of the mobility of spin labels attached to the thiol sites of membrane proteins, and it was a strong free radical scavenger in hydroxyl and superoxide anion free radical generating systems. The experimental results suggest that the presence of MTDQ in the cell membranes may improve the protection of membrane components against free radical attacks.     

Effects of metal ion chelators on DNA strand breaks and inactivation produced by hydrogen peroxide in Escherichia coli: detection of iron-independent lesions.

In order to study the role of metallic ions in the H2O2 inactivation of Escherichia coli cells, H2O2-sensitive mutants were treated with metal ion chelators and then submitted to H2O2 treatment. o-Phenanthroline, dipyridyl, desferrioxamine, and neocuproine were used as metal chelators. Cell sensitivity to H2O2 treatment was not modified by neocuproine, suggesting that copper has a minor role in OH production in E. coli. On the other hand, prior treatment with iron chelators protected the cells against the H2O2 lethal effect, indicating that iron participates in the production of OH. However, analysis of DNA sedimentation profiles and DNA degradation studies indicated that these chelators did not completely block the formation of DNA single-strand breaks by H2O2 treatment. Thiourea, a scavenger of OH, caused a reduction in both H2O2 sensitivity and DNA single-strand break production. The breaks observed after treatment with metal chelators and H2O2 were repaired 60 min after H2O2 elimination in xthA but not polA mutant cells. Therefore, we propose that there are at least two pathways for H2O2-induced DNA lesions: one produced by H2O2 through iron oxidation and OH production, in which lesions are repaired by the products of the xthA and polA genes, and the other produced by an iron-independent pathway in which DNA repair requires polA gene products but not those of the xthA gene.     

Effect of Thuya occidentalis on the labeling of red blood cells and plasma proteins with technetium-99m.

Thuya occidentalis is used in popular medicine in the treatment of condyloma and has antibacterial action. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used for several evaluations in nuclear medicine. This labeling depends on a reducing agent, usually stannous ion. Any drug which alters the labeling of the tracer could be expected to modify the disposition of the radiopharmaceutical. We have evaluated the influence of T. occidentalis extract on the labeling of RBC and plasma proteins with 99mTc. Blood was withdrawn and incubated with T. occidentalis (0.25; 2.5; 20.5; and 34.1 percent v/v). Stannous chloride (1.2 micrograms/ml) was added and then 99mTc was added. Plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. The analysis of the results shows that there is a decrease in radioactivity (from 97.64 to 75.89 percent) in BC with 34.1 percent of the drug. In the labeling process of RBC with 99mTc, the stannous and pertechnetate ions pass through the membrane, so we suggest that the T. occidentalis effect can be explained (i) by an inhibition of the transport of these ions, (ii) by damage in membrane, (iii) by competition with the cited ions for the same binding sites, or (iv) by possible generation of reactive oxygen species that could oxidize the stannous ion.     

Development of environmentally-benign wood preservatives based on the combination of organic biocides with antioxidants and metal chelators

Wood extractives can be envisaged to protect heartwood by at least three different mechanisms, i.e. fungicide, free radical scavengers/antioxidants and as metal chelators. In short-term laboratory decay tests using two different wood species and decay fungi, the combination of different organic fungicides with various antioxidants and/or metal chelators gave enhanced activity as compared to the organic biocide alone, with the best results usually obtained with all three compounds. Outdoor ground-contact stakes treated with a biocide and antioxidant combination and exposed for 30 months also gave enhanced protection against both decay fungi and termites. It was concluded that the combination of an organic biocide with metal chelating and/or antioxidant additives gives enhanced protection to wood against fungi as compared to the biocide alone and, consequently, it may be possible to develop environmentally-benign wood preservative systems based on this idea.     

Direct Regeneration of Shoots from Hairy Root Cultures of Centaurium erythraea Inoculated with Agrobacterium rhizogenes

Effect of free radical scavengers and metal chelators on polyethylene glycol (PEG, osmotic potential −1.5 MPa) induced oxidative damage in detached rice leaves was investigated. PEG treatment resulted in a decrease in relative water content and an increase in proline content, and lipid peroxidation. PEG treatment also decreased chlorophyll and protein contents. Free radical scavengers (ascorbate, sodium benzoate, reduced glutathione, and thiourea) retarded and metal chelators [2,2′-bipyridine (BP), 8-hydroxyquinoline, and 1,10-phenanthroline] prevented PEG-induced oxidative damage. Furthermore, the protective effect of BP was reversed by adding Fe²⁺ and Cu²⁺, but not by Mn²⁺ or Zn²⁺. The protective effect of BP is most likely mediated through chelation of iron. It seems that oxidative damage induced by PEG may require the participation of iron. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mechanism of protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide- and menadione-induced DNA single strand breaks in Caco-2 cells

Protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide (tert-BOOH)- and menadione-induced DNA single strand breaks was investigated in Caco-2 cells. Both tert-BOOH and menadione induced DNA single strand breaks in a concentration-dependent manner. Pre-incubation of Caco-2 cells with either quercetin or rutin for 24 h significantly decreased the formation of DNA single strand breaks evoked by tert-BOOH (P <.05). Iron chelators, 1,10-phenanthroline (o-Phen) and deferoxamine mesylate (DFO), also protected against tert-BOOH-induced DNA damage, whereas butylated hydroxytoluene (BHT) had no effect. Quercetin, and not rutin, decreased the extent of menadione-induced DNA single strand breaks. DFO and BHT, and not o-Phen, protected against menadione-induced DNA strand break formation (P <.05). From the results of this study, iron ions were involved in tert-BOOH-induced DNA single strand break formation in Caco-2 cells, whereas DNA damage evoked by menadione was far more complex. We demonstrated that the flavonoids, quercetin and rutin, protected against tert-BOOH-induced DNA strand breaks by way of their metal ion chelating mechanism. However, quercetin, and not rutin, protected against menadione-induced DNA single strand breaks by acting as both a metal chelator and radical scavenger.     

Influence of Antioxidants on Arachidonic Acid Metabolism and Platelet Function

Studies from our laboratory demonstrated that the free radical scavenger, nitro blue tetrazolium, and iron chelators, such as dypyrydil, are potent inhibitors of arachidonic acid oxidation and platelet function. In the present study, we have evaluated the effects of known antioxidants, such as butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), and diphenylamine, on arachidonic acid metabolism and platelet function. Diphenylamine, a common dye intermediate used in hair color formulations, was the most potent inhibitor of arachidonic acid metabolism by platelet cyclooxygenases. Diphenyl and BHA were also potent inhibitors of arachidonic acid oxidation. Other diphenyl analogues and BHT were relatively poor inhibitors of arachidonic-mediated platelet activation. Results of this study, as well as those of our earlier studies, suggest that antioxidants and iron chelators prevent arachidonic acid metabolism and alter platelet function by interfering with the heme/arachidonic acid interaction and blocking cyclooxygenase metabolites essential for the formation of thromboxane A₂, a potent platelet agonist.     

The role of poly(ethyleneimine) in stabilization against metal-catalyzed oxidation of proteins: a case study with lactate dehydrogenase

The protection provided by poly(ethyleneimine) (PEI) to muscle lactate dehydrogenase (LDH) in metal-catalyzed oxidation (MCO) systems (CuSO(4) or FeCl(2) combined with H(2)O(2)) was studied, and comparisons were made with the chelators EDTA and desferal, respectively. The analytical chelating capacity of PEI was estimated to be around 1 mol Cu(2+)/10 mol ethyleneimine for all molecular weights of the polymer. The effect of [PEI monomer]/[metal ion] molar ratio on the oxidatively induced aggregation of LDH exhibited a similar trend as that of the other chelators; aggregation was enhanced at lower ratios and subsequently decreased until it was undetectable with increasing ratio. In contrast, the LDH activity showed a monotonic increase with increasing concentrations of the chelator. Total protection to the enzyme by PEI was provided at concentrations lower than that needed for full chelation of the copper ions, i.e. at [PEI monomer]/[Cu(2+)] ratio above 9 in case of PEI 2000, and above 7 for PEI 25000 and 2.6 x 10(6), respectively. The polymer also provided protection against oxidation in an iron-based MCO system. Hydroxyl radical formation during the MCO reaction was inhibited in the presence of PEI. The polymer of higher molecular weights also exhibited a stronger free radical scavenging effect.     

Design, synthesis, and evaluation of novel bifunctional iron-chelators as potential agents for neuroprotection in Alzheimer's, Parkinson's, and other neurodegenerative diseases

Several novel antioxidant-iron chelators bearing 8-hydroxyoxyquinoline moiety were synthesized, and various properties related to their iron chelation, and neuroprotective action were investigated. All the chelators exhibited strong iron(III) chelating and high antioxidant properties. Chelator 9 (HLA20), having good permeability into K562 cells and moderate selective MAO-B inhibitory activity (IC50 110 microM), displayed the hightest protective effects against differentiated P19 cell death induced by 6-hydroxydopamine. EPR studies suggested that Chelator 9 also act as radical scavenger to directly scavenge hydroxyl radical.     

Probing determinants of the metal ion selectivity in carbonic anhydrase using mutagenesis

Few studies measuring thermodynamic metal ion selectivity of metalloproteins have been performed, and the major determinants of metal ion selectivity in proteins are not yet well understood. Several features of metal ion binding sites and metal coordination have been hypothesized to alter the transition metal selectivity of chelators, including (1) the polarizability of the coordinating atom, (2) the relative sizes of the binding site and the metal ion, and (3) the metal ion binding site geometry. To test these hypotheses, we have measured the metal ion affinity and selectivity of a prototypical zinc enzyme, human carbonic anhydrase II (CAII), and a number of active site variants where one of the coordinating ligands is substituted by another side chain capable of coordinating metal. CAII and almost all of the variants follow the inherent metal ion affinity trend suggested by the Irving-Williams series, demonstrating that this trend operates within proteins as well as within small molecule chelators and may be a dominant factor in metal ion selectivity in biology. Neither the polarizability of the liganding side chains nor the size of the metal ion binding site correlates strongly with metal ion specificity; instead, changes in metal ion specificity in the variants correlate with the preferred coordination number and geometry of the metal ion. This correlation suggests that a primary feature driving deviations from the inherent ligand affinity trend is the positioning of active site groups such that a given metal ion can adopt a preferred coordination number/geometry.     

Effect of eggplant (Solanum melongena) extract on the in vitro labeling of blood elements with technetium-99m and on the biodistribution of sodium pertechnetate in rats.

The use of eggplant has been suggested to treat different diseases. We studied the effect of eggplant extract on the labeling of red blood cells (RBC) and plasma proteins with technetium-99m (Tc-99m) and on biodistribution of sodium pertechnetate (Tc-99m) in rats. Blood was incubated with an eggplant extract (final concentrations 3.12 to 250.00 mg/ml) for 60 min. Then, stannous chloride (SnCl2) (0.06 or 1.2 microg/ml) and Tc-99m, as sodium pertechnetate, were added. Samples of RBC and plasma (P) were separated and also precipitated and soluble (SF) and insoluble (IF) fractions were isolated. The percent of radioactivity (%ATI) in the fractions was calculated. In the biodistribution study, Wistar rats were treated with eggplant extract (300 mg/ml) for 4 weeks, in drinking water. Tc-99m was administered in the rats, after 90 min they were sacrificed and organs and blood were isolated. When 0.06 microg/ml SnCl2 was used, eggplant extract: i/ inhibited the label of RBC (97.14 +/- 2.01 to 52.21 +/- 3.97%ATI), ii/ decreased the labeling in IF-P from 38.79 +/- 11.73 to 5.49 +/- 2.65%ATI, and iii/ diminished the labeling in IF-RBC from 90.04 +/- 2.65 to 46.17 +/- 9.49%ATI. This inhibitory effect was not observed with SnCl2 1.2 microg/ml. In the biodistribution study, the %ATI: i/ increased in the liver from 2.15 +/- 0.54 to 3.11 +/- 1.29 and ii/ in the other organs the Tc-99m uptake was not modified. The uptake of Tc-99m in red blood cells protein (IF-RBC) decreased from 66.62 +/- 19.67 to 31.66 +/- 8.84%. It is possible to suggest that some components of the eggplant extract present an oxidation power able to alter the fixation of the Tc-99m on the blood elements. Moreover, as eggplant is metabolized in the liver, this fact could justify the alteration of the uptake in this organ.     

The Bactericidal Action of Peroxides; An E.P.R. Spin-Trapping Study

E.P.R. spin trapping has been employed to study radical production during the bactericidal action of three peroxide compounds (peracetic acid, 4-percarboxy-N-isobutyltrimellitimide and magnesium monoperoxyphthalate) upon both Gram negative (Escherichia Coll) and Gram positive (Staphylococcus Aureus) bacteria. Use of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has allowed direct detection of both carbon-centred and hydroxyl radicals, which are produced at varying rates for the different bacteria/peracid systems studied. The inhibition of bactericidal action, by DMPO and two antioxidants, Vitamin C and Trolox C, indicates that radicals are the lethal species and evidence is presented which suggests that radical production is internal to the bacterial cell. Hydroxyl radicals are believed to be the lethal species. The effect of added iron chelators and haem protein inhibitors indicates that iron species and haem proteins in particular are involved. A marked variation is found in observed hydroxyl-radical adduct signals with both the nature and concentration of peracid. A strong inverse correlation is found between the concentration of the observed radical adduct signal and the relative strength of the peroxide as a bactericide; use of a stable nitroxide as a radical scavenger confirms that strong bactericides produce radicals at a much faster rate than weak bactericides. Plots of radical generation versus time are correlated with % bacterial kill, offering further evidence that hydroxyl radicals are the lethal species.     

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: oxidative damage by hydroxyl free radicals and its prevention

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.     

Roles of copper and O(2) in the radiation-induced inactivation of T7 bacteriophage

The effect of copper on the radiation damage induced in T7 bacteriophage has been investigated. The phages were gamma-irradiated and the effects of copper(II) ions in the presence of various additives and radical scavengers were examined in an attempt to better understand the effect of transition metal ions on the role of free radicals, particularly superoxide, in biological damage. The present work extends a study previously done on isolated enzyme to a whole biological entity. Copper(II) ions even at very low concentrations enhanced the lethal effect of radiation. This sensitization was observed in both the presence and the absence of oxygen. The effect of copper could be reverted by chelating agents such as EDTA or 1,10-phenanthroline. Hydrogen peroxide enhanced the sensitizing effect of copper, though little if any protection was provided by catalase or SOD. High molecular weight scavengers of free radicals in the presence of both copper(II) and hydrogen peroxide had no protective effect. (This is in contrast to metal-free systems where, although such scavengers are incapable of penetrating the phages, they protect them against inactivation.) These scavengers, without added H2O2, afforded only slight protection to the irradiated phages in the presence of Cu. Low molecular weight scavengers of free radicals reduced but did not eliminate the sensitizing effect of copper. The sensitizing effect of copper was also observed with other T-odd phages, but not with the T-even series. Copper(II) ions under similar experimental conditions did not sensitize T4 or T2 phages but rather had a protective effect. The results are interpreted in terms of a site-specific Fenton mechanism according to which the binding of the metal ion to the phages is a prerequisite for the occurrence of the biological damage. The results also indicate that most of the copper effect is endogenous. This is in accord with the failure of copper to sensitize the T-even phages, which differ by the rigidity and permeability of their outer coat structures.     

Adaptive response to H(2)O(2) protects against SnCl(2) damage: the OxyR system involvement

The stannous ion, mainly the stannous chloride (SnCl(2)) salt form, is widely used as a reducing agent to label radiotracers with technetium-99m ((99m)Tc). These radiotracers can be employed as radiopharmaceuticals in nuclear medicine procedures. In this case, there is no doubt about absorption of this complex, because it is intravenously administered in humans, although biological effects of these agents have not been fully understood. In this work we used a bacterial system to study the cytotoxic potential of stannous chloride. It is known that SnCl(2) induces lesions that could be mediated by reactive oxygen species (ROS). We, thus, investigated the existence of cross-adaptive response between hydrogen peroxide (H(2)O(2)) and SnCl(2) and the role of the OxyR system known to promote cellular protection against oxidative damages. Here we describe the results obtained with prior treatment of different Escherichia coli strains with sub-lethal doses of H(2)O(2), followed by incubation with SnCl(2). Our data show that H(2)O(2) is capable of inducing cross-adaptive response against the lethality promoted by SnCl(2), suggesting the OxyR system participation through catalase, alkyl hydroperoxide reductase and superoxide dismutase enzymes     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery. Failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, 99mTc, and an aqueous marker, 125I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped 125I-labelled poly(vinyl pyrrolidone) were labelled with 99mTc by the SnCl2 method. 99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than 125I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, 99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than 125I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60-70% of 125I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of 99mTc-radioactivity was co-eluted with the liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all 99mTc-radioactivity does not represent the 60-70% of intact liposomes present in lymph nodes. Using the aqueous marker 125I-labelled poly(vinyl pyrrolidone), the lymph node localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that 125I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier, where the authors used 99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became 'leaky' to low-molecular-weight drugs, for example, methotrexate (Mr 454) to a much greater extent than with a large-molecular-weight substance, 125I-labelled poly(vinyl pyrrolidone) (Mr 30 000-40 000), when incubated with rat lymph at 37 degrees C. Using the two markers 99mTc and 125I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining lambda-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with 99mTc rather than using the SnCl2 technique, or using other radionuclides as markers for gamma-scan imaging.     

Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants

Cupric ions (Cu2+) and ferric ions (Fe3+) added to hydrogen peroxide generate hydroxyl radicals (OH) capable of degrading deoxyribose with the formation of thiobarbituric acid-reactive products. This damage can be inhibited by catalase, OH radical scavengers and specific metal ion chelators. All proteins tested nonspecifically inhibited copper-dependent damage but have little effect on the iron-dependent reaction. Copper ions appear to bind to the proteins which prevents formation of OH radicals in free solution. However, OH radicals are still generated at a site-specific location on the protein molecule. Protein damage is detected as fluorescent changes in amino acid residues.