Janice A. Lumpkin

Hyperoxia induces DNA damage in mammalian cells

There is mounting evidence on the role of oxygen-derived free radicals in causing damage to various cellular components. However, most studies reported in the literature have been conducted under conditions where cells were challenged with chemical free radical generating systems. In contrast, we measured DNA strand breaks, through a relatively simple and sensitive technique, as a function of the dissolved oxygen tension in a bioreactor. Cells were exposed to a step change in oxygen tension at mid-exponential growth phase. Several levels of oxygen were tested (200, 300, and 476% dissolved oxygen with respect to air saturation at 1 atmosphere) and compared against a control (10% dissolved oxygen). Hyperoxia was found to cause monotonically increasing DNA strand breakage at all the oxygen levels. In addition, hyperoxia was found to affect other metabolic functions such as the glucose consumption rate, lactate production rate, and cell growth. When hyperoxia-induced DNA strand breakage was compared to that induced by exposure to hydrogen peroxide, a similar response was observed. Exposure to a dissolved oxygen level of 200% induced DNA strand breakage comparable to a bolus of 4.2 microM hydrogen peroxide. Our results show that there is an association between hyperoxia and DNA damage.

DNA base modifications and membrane damage in cultured mammalian cells treated with iron ions

We investigated DNA base damage in mammalian cells exposed to exogenous iron ions in culture. Murine hybridoma cells were treated with Fe(II) ions at concentrations of 10 microM, 100 microM, and 1 mM. Chromatin was isolated from treated and control cells and analyzed by gas chromatography/mass spectrometry for DNA base damage. Ten modified DNA bases were identified in both Fe(II)-treated and control cells. The quantification of modified bases was achieved by isotope-dilution mass spectrometry. In Fe(II)-treated cells, the amounts of modified bases were increased significantly above the background levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M in the culture medium did not significantly inhibit the formation of modified DNA bases. A mathematical simulation used to evaluate the plausibility of DNA damage upon Fe(II) treatment predicted a dose-dependent response, which agreed with the experimental results. In addition, Fe(II) treatment of cells increased the cell membrane permeability and caused production of lipid peroxides. The nature of DNA base lesions suggests the involvement of the hydroxyl radical in their formation. The failure of dimethyl sulfoxide to inhibit their formation indicates a site-specific mechanism for DNA damage with involvement of DNA-bound metal ions. Fe(II) treatment of cells may increase the intracellular iron ion concentration and/or cause oxidative stress releasing metal ions from their storage sites with subsequent binding to DNA. Identified DNA base lesions may be promutagenic and play a role in pathologic processes associated with iron ions.

Tert.-Butyl hydroperoxide-mediated DNA base damage in cultured mammalian cells

tert.-Butyl hydroperoxide has been utilized to study the effect of oxidative stress on living cells; however, its effect on DNA bases in cells has not been characterized. In the present work, we have investigated DNA base damage in mammalian cells exposed to this organic hydroperoxide. SP2/0 derived murine hybridoma cells were treated with 4 concentrations of tert.-butyl hydroperoxide for varying periods of time. Chromatin was isolated from treated and control cells and subsequently analyzed by gas chromatography-mass spectrometry with selected-ion monitoring for DNA base damage. Quantification of damaged DNA bases was achieved by isotope-dilution mass spectrometry. The amounts of 8 products were significantly higher than control levels in cells treated with tert.-butyl hydroperoxide at a concentration range of 0.01-0.1 mM. At concentrations from 1.0 to 10 mM, product formation was inhibited and the amounts of products were similar to those in control cells. The bimodal nature of the dose-response may be qualitatively analogous to previous reports of bimodal killing of E. coli bacteria by hydrogen peroxide. The nature of the identified DNA base lesions suggests the involvement of the hydroxyl radical in their formation. tert.-Butyl hydroperoxide is known to produce the tert.-butoxyl radical in reactions with metal ions. However, it is unlikely that the tert.-butoxyl radical produces these DNA lesions. It is suggested that DNA base damage arises from tert.-butyl hydroperoxide-mediated oxidative stress in cells, resulting in formation of hydroxyl radicals in close proximity to DNA. The inhibition of product formation at high concentrations of tert.-butyl hydroperoxide may be explained by the scavenging of tert.-butoxyl radical by tert.-butyl hydroperoxide resulting in inhibition of oxidative stress. The plausibility of the scavenging mechanism was evaluated with a mathematical simulation of the dose-response for DNA damage in solutions containing hydrogen peroxide. The simulation model predicted a bimodal dose-response which agreed qualitatively with the results in this study and with other in vivo and in vitro studies reported in the literature.

Inactivation of lysozyme by sonication under conditions relevant to microencapsulation.

Controlled release dosage forms of proteins and other biomolecules can be prepared by microencapsulating them in polymeric microspheres. Proteins are subjected to potentially damaging effects of sonication and exposure to organic solvents during the microencapsulation process. The relatively stable enzyme lysozyme was dissolved in aqueous buffer and sonicated in the presence of methylene chloride to mimic the initial step of the microencapsulation process. The stability of lysozyme was evaluated by determining the enzyme activity before and after sonication, size-exclusion chromatography, native polyacrylamide gel electrophoresis, and by measuring the amount of precipitates formed. Following sonication, the total protein introduced was distributed between a soluble and an insoluble fraction. Sonication of lysozyme solutions in the presence of methylene chloride led to an increase in precipitates. The precipitates were enzymatically inactive, did not dissolve easily, and were held by non-covalent interactions. No fragments or aggregates of lysozyme were detectable in the soluble fraction. Sonicating aqueous lysozyme solutions with and without methylene chloride decreased the specific activity of the enzyme in the soluble fraction. Excipients such as dimethyl sulfoxide (DMSO), mannitol, sucrose, and tween 80 were included in the sonication mixtures containing lysozyme. With the exception of tween 80, the addition of the excipients to aqueous solutions of lysozyme led to a greater decrease in the specific activity of lysozyme when sonicated in the presence of methylene chloride. DMSO caused the greatest loss of enzyme activity following sonication. Sonication of lysozyme with water, methylene chloride, and DMSO yielded methyl radicals, which were trapped with alpha-phenyl N-tert-butylnitrone and detected by ESR spectroscopy.

Mathematical modeling of drug release from hydrogel matrices via a diffusion coupled with desorption mechanism

Abstract Model calculations have been performed to account for the kinetics of drug release from polymeric matrices where both diffusion and desorption mechanisms control the overall release rate. The model incorporates both a desorption term, based on Langmuir kinetics, and a transient Fickian diffusion term and is solved for infinite sink boundary conditions. Solutions by both finite difference and finite element techniques indicate the effects of binding interaction strength, binding capacity, amount of drug loaded and rate of desorption on the release kinetics of drugs adsorbed to polymer matrices. Simulation results indicate that it is possible to modulate the net release rates of drugs over a wide range by altering the binding parameters enumerated above as well as drug diffusivities in the matrix.

The effect of electrostatic charge interactions on release rates of gentamicin from collagen matrices.

AbstractPurpose. This work studied the effect of changes in the magnitude of electrostatic charge interactions on the release kinetics of gentamicin from collagen matrices. Methods. The charge distribution on collagen was altered by specific charge chemistries to yield net negative charges which exhibited binding interactions with positively charged gentamicin. The adsorption isotherms were measured to characterize binding interactions and release of gentamicin from modified matrices were measured. The release rates were compared to a mathematical model based on an instantaneous desorption coupled with diffusion mechanism. Results. Ninety percent of the gentamicin loaded was released from native collagen matrices in 2.5 days (one-sided slab geometry in-vitro). Succinylated collagen matrices released 70% in 2.5 days and phosphonylated collagen matrices released 50% in 2.5 days. Excellent agreement between model predictions and experiment results were obtained. Conclusions. Modified collagen can be much more effective in antibiotic therapy in sustaining release rates compared to native collagen for charged antibiotics like gentamicin.

Effect of electrostatic interactions on polylysine release rates from collagen matrices and comparison with model predictions

Collagen was investigated as a drug delivery matrix because it is injectable, biocompatible and is naturally resorbed in the body. This work focuses on understanding the effect of electrostatically mediated binding interactions on release kinetics of charged polypeptides from collagen matrices. The charge distribution on collagen molecules can be modified by specific chemical reactions to yield net negative or net positive charges at physiological pH. These electrostatic charges are favorable in binding oppositely charged polypeptides. An ESR based technique was utilized to measure adsorption isotherms in order to characterize the strength of binding interactions. Adsorption isotherm measurements indicate that increasing the charge density of collagen enhances binding interactions. Enhanced binding interactions are also observed when the degree of charge modification is increased as well as when the prevailing ionic strength of the matrix is lowered. Charged polypeptides show slower release rates from higher charge density collagen matrices. Release kinetics have been modeled via coupled desorption and diffusion mechanisms and comparisons based on independent measurements of desorption (kinetic and equilibrium) and diffusional parameters with experimentally measured release rates are in good agreement.

Structural damage to lactate dehydrogenase during copper iminodiacetic acid metal affinity chromatography.

The stability of the enzyme lactate dehydrogenase (LDH) was evaluated by measuring structural damage and activity loss after exposure to copper−iminodiacetic acid (IDA) immobilized metal affinity chromatography (IMAC) under oxidizing conditions at pH 7.0. Oxidizing conditions were produced by adding reductants commonly employed in bioprocessing and biomedical applications (glutathione, β‐mercaptoethanol, dithiothreitol, cysteine, or ascorbate) and/or hydrogen peroxide to the mobile phase. Most of these additives have been shown recently to give rise to metal‐catalyzed oxidation (MCO) reactions on copper−iminodicaetic acid IMAC columns. Structural damage in the form of increased susceptibility to proteolytic degradation, fragmentation, and cross‐linking were measured. Increased sensitivity to proteolysis was significant in virtually all cases tested, even when activity remained high (>95% specific activity recovered). In contrast fragmentation and cross‐linking were minimal in all cases, even when activity was low (<50%). As the damage was believed to have been caused primarily by MCO reactions, preventative measures consistent with this reaction pathway were tested. The most successful measure for all of the conditions studied was addition of the Cu+ chelating agent bicinchoninic acid (BCA) to the mobile phase. Decreased contact time with the column decreased damage in the case where glutathione was added. Removal of dissolved oxygen by nitrogen sparging and use of Tris−acetate buffer in place of phosphate had no measurable effect. The success of BCA addition in reducing structural damage and activity loss strengthens the conclusion that MCO reactions can occur on copper−iminodiacetic acid IMAC columns. However, the addition of BCA and the other protective measures described were not successful in eliminating the increased proteolytic susceptibility observed when LDH in buffer was exposed to the copper‐charged column with no oxidizing additives. This suggests that at least one other pathway for damage exists. This damage is difficult to detect as it did not cause statistically significant losses in enzymatic activity, fragmentation, or cross‐linking.

Tert-butyl hydroperoxide-mediated DNA base damage in cultured mammalian cells

tert.-Butyl hydroperoxide has been utilized to study the effect of oxidative stress on living cells; however, its effect on DNA bases in cells has not been characterized. In the present work, we have investigated DNA base damage in mammalian cells exposed to this organic hydroperoxide. SP2/0 derived murine hybridoma cells were treated with 4 concentrations of tert.-butyl hydroperoxide for varying periods of time. Chromatin was isolated from treated and control cells and subsequently analyzed by gas chromatography-mass spectrometry with selected-ion monitoring for DNA base damage. Quantification of damaged DNA bases was achieved by isotope-dilution mass spectrometry. The amounts of 8 products were significantly higher than control levels in cells treated with tert.-butyl hydroperoxide at a concentration range of 0.01-0.1 mM. At concentrations from 1.0 to 10 mM, product formation was inhibited and the amounts of products were similar to those in control cells. The bimodal nature of the dose-response may be qualitatively analogous to previous reports of bimodal killing of E. coli bacteria by hydrogen peroxide. The nature of the identified DNA base lesions suggests the involvement of the hydroxyl radical in their formation. tert.-Butyl hydroperoxide is known to produce the tert.-butoxyl radical in reactions with metal ions. However, it is unlikely that the tert.-butoxyl radical produces these DNA lesions. It is suggested that DNA base damage arises from tert.-butyl hydroperoxide-mediated oxidative stress in cells, resulting in formation of hydroxyl radicals in close proximity to DNA. The inhibition of product formation at high concentrations of tert.-butyl hydroperoxide may be explained by the scavenging of tert.-butoxyl radical by tert.-butyl hydroperoxide resulting in inhibition of oxidative stress. The plausibility of the scavenging mechanism was evaluated with a mathematical simulation of the dose-response for DNA damage in solutions containing hydrogen peroxide. The simulation model predicted a bimodal dose-response which agreed qualitatively with the results in this study and with other in vivo and in vitro studies reported in the literature.