Metal contaminants promote degradation of lipid/DNA complexes during lyophilization

Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe²⁺) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.     

Degradation of lyophilized lipid/DNA complexes during storage: The role of lipid and reactive oxygen species

The presence of trace amounts of metal ions in nonviral vector formulations can significantly affect the stability of lipid/DNA complexes (lipoplexes) during acute freeze-drying. The goal of the present study was to evaluate the generation of reactive oxygen species (ROS) in dried formulations of lipoplexes and in their individual components (lipid or naked DNA). The experiments were conducted in the presence or absence of a transition metal (Fe²⁺). Lipoplexes and their individual components were formulated in trehalose and subjected to lyophilization and stored for a period of up to 2 months at + 60 °C. Physico-chemical characteristics and biological activity were evaluated at different time intervals. Generation of ROS during storage was determined by adding a fluorescence probe to the formulations prior to freeze-drying. We also monitored the formation of thiobarbituric reactive substances (TBARS). Our results show that ROS and TBARS form during storage in the dried state. Our findings also suggest that degradation is more rapid in the presence of lipid, even in the absence of metal. We also showed that dried naked DNA formulations are more stable without the lipid component. Effective strategies are then needed to minimize the formation and accumulation of oxidative damage of lipoplexes during storage.     

Mixed chelate copper complex, Casiopeina IIgly, binds and degrades nucleic acids: a mechanism of cytotoxicity

Metal-containing drugs that interact with DNA have been designed and studied for their anticancer activity. In this study, the mixed chelate copper-based anticancer drugs, the casiopeinas, were found to bind to DNA and to degrade DNA and RNA in the presence of reducing agents (e.g. ascorbic acid). Casiopeinas binding to DNA is high affinity, with harsh wash conditions failing to remove the interaction. The reaction requires oxygen, probably involved in the generation of *OH radicals, which would be responsible for the strand breakage. The reaction was diminished by catalase, and was completely abolished by copper chelators (e.g. trientine, EDTA); however, superoxide dismutase (SOD) had no significant effect on casiopeina-mediated DNA degradation. Casiopeina IIgly (casIIgly) in the presence of ascorbate was capable of degrading RNA, plasmid and genomic DNA, and chromatin and intranuclear genetic material. Moreover, catalase and/or SOD partially protected cells, ascorbic acid enhanced and trientine, a copper chelator, abolished the cytotoxicity of casIIgly. The generation of 8-oxodG in cells exposed to casIIgly suggests that the generation of ROS is the major cause of the cytotoxicity observed and underlies the high toxicity and anticancer activity of these compounds.     

Cellular responses to reactive oxygen species-induced DNA damage and aging

Oxidative stress in cells and tissues can occur during pathophysiological developments, e.g., during inflammatory and allergic diseases or during ischemic or toxic and hyperglycemic conditions via the generation of reactive oxygen species (ROS). Moreover, ROS can be generated by radiation (UV, X-rays) and pharmacologically, e.g., by anthracyclins as chemotherapeutic compounds for treatment of a variety of tumors to induce 'stress or aberrant signaling-inducing senescence' (STASIS). Although STASIS is distinguished from intracellular replicative senescence, a variety of cellular mechanisms appear similar in both aging pathways. It is generally accepted that oxidative stress and ROS eventually cause DNA damage, whereby insufficient cellular repair mechanisms may contribute to premature aging and apoptosis. Conversely, ROS-induced imbalances of the signaling pathways for metabolic protein turnover may also result in opposite effects to recruit malfunctioning aberrant proteins and compounds that trigger tumorigenic processes. Consequently, DNA damage plays a role in the development of carcinogenesis, but is also associated with an aging process in cells and organisms.     

Alpha-synuclein and the pathogenesis of Parkinson's disease

Lesions known as Lewy bodies (LBs) and Lewy neurites (LNs) characterise brains of Parkinson's disease (PD) patients. Intracellular aggregation of alpha-synuclein (alpha-syn) appears to play a key role in the generation of LBs and LNs. Such aggregation in the presence of redox metals may initiate Fenton reaction-mediated generation of reactive oxygen species (ROS). ROS thus generated may result in cytotoxic mechanisms such as the induction of DNA single-strand breaks.     

Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: the particle isolation hypothesis

The instability of nonviral vectors in aqueous suspensions has stimulated an interest in developing lyophilized formulations for use in gene therapy. Previous work has demonstrated a strong correlation between the maintenance of particle size and retention of transfection rates. Our earlier work has shown that aggregation of nonviral vectors typically occurs during the freezing step of the lyophilization process, and that high concentrations of sugars are capable of maintaining particle size. This study extends these observations, and demonstrates that glass formation is not the mechanism by which sugars protect lipid/DNA complexes during freezing. We also show that polymers (e.g., hydroxyethyl starch) are not capable of preventing aggregation despite their ability to form glasses at relatively high subzero temperatures. Instead, our data suggest that it is the separation of individual particles within the unfrozen fraction that prevents aggregation during freezing, i.e., the particle isolation hypothesis. Furthermore, we suggest that the relatively low surface tension of mono- and disaccharides, as compared to starch, allows phase-separated particles to remain dispersed within the unfrozen excipient solution, which preserves particle size and transfection rates during freezing.     

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.     

Oxidative DNA damage induced by HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer in the presence of Au(III)

Oxidative DNA damage was investigated by free radicals generated from HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer, which is widely used in biochemical or biological studies, in the presence of Au(III). The effect of free radicals on the DNA damage was ascertained by gel electrophoresis, electron spin resonance (ESR) spectroscopy and circular dichroism (CD) spectroscopy. ESR results indicated the generation of nitrogen-centered cationic free radicals from the HEPES in the presence of Au(III) which cause the DNA damage. No ESR spectra were observed for phosphate, tris(hydroxymethyl)aminomethane (Tris-HCl) and acetate buffers in the presence of Au(III) or for HEPES buffer in the presence of other metal ions such as Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II) or [Au(III)(TMPyP)](5+) and [Pd(II)(TMPyP)](4+), where [H(2)(TMPyP)](4+) denotes tetrakis(1-methylpyridium-4-yl)porphyrin. Consequently, no DNA damage was observed for these buffer agents (e.g., phosphate, Tris-HCl or acetate) in the presence of Au(III) or for HEPES in the presence of other metal ions or the metalloporphyrins mentioned above. No detectable inhibitory effect on the DNA damage was observed by using the typical scavengers of reactive oxygen species (ROS) ()OH, O(2)(-) and H(2)O(2). This non-inhibitory effect indicated that no reactive oxygen species were generated during the incubation of DNA with HEPES and Au(III). The drastic change in CD spectra from positive ellipticity to negative ellipticity approximately at 270 nm with increasing concentration of Au(III) also indicated the significant damage of DNA. Only HEPES or Au(III) itself did not damage DNA. A mechanism for the damaging of DNA is proposed.     

Tuning Cell Cycle Regulation with an Iron Key

Iron (Fe) is essential for cellular metabolism e.g., DNA synthesis and its depletion causes G1/S arrest and apoptosis. Considering this, Fe chelators have been shown to be effective anti-proliferative agents. In order to understand the anti-tumor activity of Fe chelators, the mechanisms responsible for G1/S arrest and apoptosis after Fe-depletion have been investigated. These studies reveal a multitude of cell cycle control molecules are regulated by Fe. These include p53, p27Kip1, cyclin D1 and cyclin-dependent kinase 2 (cdk2). Additionally, Fe-depletion up-regulates the mRNA levels of the cdk inhibitor, p21CIP1/WAF1, but paradoxically down-regulates its protein expression. This effect could contribute to the apoptosis observed after Fe-depletion. Iron-depletion also leads to proteasomal degradation of p21CIP1/WAF1 and cyclin D1 via an ubiquitin-independent pathway. This is in contrast to the mechanism in Fe-replete cells, where it occurs by ubiquitin-dependent proteasomal degradation. Up-regulation of p38 mitogen-activated protein kinase (MAPK) after Fe-depletion suggests another facet of cell cycle regulation responsible for inhibition of proliferation and apoptosis induction. Elucidation of the complex effects of Fe-depletion on the expression of cell cycle control molecules remains at its infancy. However, these processes are important to dissect for complete understanding of Fe-deficiency and the development of chelators for cancer treatment.     

Iron-catalyzed reactions may be responsible for the biochemical and biological effects of asbestos

The most carcinogenic forms of asbestos contain iron to levels as high as 36% by weight and catalyze many of the same biochemical reactions that freshly prepared solutions of iron do, i.e. oxygen consumption, generation of reactive oxygen species, lipid peroxidation and DNA damage. The participation of iron from asbestos in these reactions has been demonstrated using the iron chelator desferrioxamine B which inhibits iron-catalyzed reactions. Iron appears to be redox active on the asbestos fiber, but chelation and subsequent iron mobilization from asbestos by a variety of chelators, e.g. citrate, EDTA or nitrilotriacetate, makes the iron more redox active resulting in greater oxygen consumption and production of oxygen radicals in the presence of reducing agents. Iron also appears to be important for some of the asbestos-dependent biological effects on tissues or cells in culture, such as phagocytosis, cytotoxicity, lipid peroxidation and DNA damage. Therefore, redox cycling of iron to generate oxygen radicals at the surface of the fiber and/or in solution, as mobilized, low molecular weight chelates, may be very important in eliciting some of the biological effects of asbestos in vivo.     

Effect of chelating agents and metal ions on the degradation of DNA by bleomycin.

The degradation of DNA by bleomycin was studied in the absence and in the presence of added reducing agents, including 2-mercaptoethanol, dithiothreitol, reduced nicotinamide adenine dinucleotide phosphate, H2O2, and ascorbate, and in the presence of a superoxide anion generating system consisting of xanthine oxidase and hypoxanthine. In all cases, breakage of DNA was inhibited by low concentrations of chelators; where examined in detail, deferoxamine mesylate was considerably more potent than (ethylenedinitrilo)tetraacetic acid. Iron was found to be present in significant quantities in all reaction mixtures. Thus, the pattern of inhibition observed is attributed to the involvement of contaminating iron in the degradation of DNA by bleomycin. Cu(II), Zn(II), and Co(II) inhibit degradation of DNA by bleomycin and Fe(II) in the absence of added reducing agents. A model is proposed in which the degradation of DNA in these systems is dependent on the oxidation of an Fe(II)-bleomycin-DNA complex.     

Intermediates of replication of NR1 plasmid DNA in Escherichia coli mini-cells

The pulse label of NRL plasmid-containing mini-cells has been shown to be localized mainly in DNA with a floating density in the CsCl-EtBr gradient different from the floating density of supercoil and open circle DNAs. During the chase of the pulse label, the DNA is transfered from the fraction with the intermediate floating density varying between the values for the supercoil and open circle DNA fractions to the fraction located below supercoil DNA in the equilibrium gradient and further to the open circle fraction. Electron microscopic analysis of the material with a higher floating density as compared to supercoil DNA has demonstrated the presence of "heavy" intermediates--covalently closed loosely supercoiled molecules. It is also supported by the sedimentation pattern of the characterized fraction in neutral and alkaline saccharose gradients. Molecules located in the CsCl-EtBr gradient between supercoil and open circle DNAs have the sedimentation constant characteristic for the elongation intermediates. It is suggested that NRL DNA molecules in E. coli mini-cells pass through all the basic stages of replication which results in the formation of open circle DNA or supercoil relaxation complexes.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Topical applications of iron chelators in photosensitization.

Generation of the reactive oxygen species (ROS) in skin by exposure to ultraviolet (UV) radiation induces a number of cutaneous pathologies such as skin cancer, photosensitization, and photoaging among others. Skin iron catalyzes UV generation of ROS. Topical application of iron chelators reduces erythema, epidermal and dermal hypertrophy, wrinkle formation, tumour appearance. It has been proposed that iron chelators can be useful agents against damaging effects of both short- and long-term UV exposure. A better understanding of the action mechanisms of iron chelators, might be useful to developing effective anticancer and antiphotoaging cosmetic products. Iron chelators may lead to accumulation of protoporphyrin IX (PpIX), a strong photosensitizer. The action of iron chelators in skin, related to PpIX increase has not yet been thoroughly studied. Therefore, we have investigated the formation of PpIX in normal mouse skin after topical application of creams containing metal chelators. The amount and distribution of porphyrins formed was determined by means of non-invasive fluorescence spectroscopy. Deferoxamine (DF), ethylenediaminetetraacetic acid (EDTA), 1,2-diethyl-3-hydroxypyridin-4-one (CP94), but not meso-2,3-dimercaptosuccinic acid (DMSA), caused increased accumulation of endogenous porphyrins in the skin. Fluorescence excitation and emission spectroscopy confirmed that PpIX was the main fluorescent species. The amount of PpIX accumulated in skin under the present conditions was not large enough to produce any significant erythema after light exposure. Further studies are needed to evaluate the role of PpIX induced by iron chelators used, against photoaging and cancer prevention.     

Cell-penetrating peptides: breaking through to the other side

Cell-penetrating peptides (CPPs) have been previously shown to be powerful transport vector tools for the intracellular delivery of a large variety of cargoes through the cell membrane. Intracellular delivery of plasmid DNA (pDNA), oligonucleotides, small interfering RNAs (siRNAs), proteins and peptides, contrast agents, drugs, as well as various nanoparticulate pharmaceutical carriers (e.g., liposomes, micelles) has been demonstrated both in vitro and in vivo. This review focuses on the peptide-based strategy for intracellular delivery of CPP-modified nanocarriers to deliver small molecule drugs or DNA. In addition, we discuss the rationales for the design of 'smart' pharmaceutical nanocarriers in which the cell-penetrating properties are hidden until triggered by exposure to appropriate environmental conditions (e.g., a particular pH, temperature, or enzyme level), applied local microwave, ultrasound, or radiofrequency radiation.     

Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism

To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach.     

Oxidative stress and metal carcinogenesis

Occupational and environmental exposures to metals are closely associated with an increased risk of various cancers. Although carcinogenesis caused by metals has been intensively investigated, the exact mechanisms of action are still unclear. Accumulating evidence indicates that reactive oxygen species (ROS) generated by metals play important roles in the etiology of degenerative and chronic diseases. This review covers recent advances in (1) metal-induced generation of ROS and the related mechanisms; (2) the relationship between metal-mediated ROS generation and carcinogenesis; and (3) the signaling proteins involved in metal-induced carcinogenesis, especially intracellular reduction-oxidation-sensitive molecules.