Radiation epidemiology and health effects following low-level radiation exposure.

Radiation epidemiology is the study of human disease following radiation exposure to populations. Epidemiologic studies of radiation-exposed populations have been conducted for nearly 100 years, starting with the radium dial painters in the 1920s and most recently with large-scale studies of radiation workers. As radiation epidemiology has become increasingly sophisticated it is used for setting radiation protection standards as well as to guide the compensation programmes in place for nuclear weapons workers, nuclear weapons test participants, and other occupationally exposed workers in the United States and elsewhere. It is known with high assurance that radiation effects at levels above 100-150 mGy can be detected as evidenced in multiple population studies conducted around the world. The challenge for radiation epidemiology is evaluating the effects at low doses, below about 100 mGy of low-linear energy transfer radiation, and assessing the risks following low dose-rate exposures over years. The weakness of radiation epidemiology in directly studying low dose and low dose-rate exposures is that the signal, i.e. the excess numbers of cancers associated with low-level radiation exposure, is so very small that it cannot be seen against the very high background occurrence of cancer in the population, i.e. a lifetime risk of incidence reaching up to about 38% (i.e. 1 in 3 persons will develop a cancer in their lifetime). Thus, extrapolation models are used for the management of risk at low doses and low dose rates, but having adequate information from low dose and low dose-rate studies would be highly desirable. An overview of recently conducted radiation epidemiologic studies which evaluate risk following low-level radiation exposures is presented. Future improvements in risk assessment for radiation protection may come from increasingly informative epidemiologic studies, combined with mechanistic radiobiologic understanding of adverse outcome pathways, with both incorporated into biologically based models.

TOPAS-nBio: An Extension to the TOPAS Simulation Toolkit for Cellular and Sub-cellular Radiobiology.

The TOPAS Monte Carlo (MC) system is used in radiation therapy and medical imaging research, having played a significant role in making Monte Carlo simulations widely available for proton therapy related research. While TOPAS provides detailed simulations of patient scale properties, the fundamental unit of the biological response to radiation is a cell. Thus, our goal was to develop TOPAS-nBio, an extension of TOPAS dedicated to advance understanding of radiobiological effects at the (sub-)cellular, (i.e., the cellular and sub-cellular) scale. TOPAS-nBio was designed as a set of open source classes that extends TOPAS to model radiobiological experiments. TOPAS-nBio is based on and extends Geant4-DNA, which extends the Geant4 toolkit, the basis of TOPAS, to include very low-energy interactions of particles down to vibrational energies, explicitly simulates every particle interaction (i.e., without using condensed histories) and propagates radiolysis products. To further facilitate the use of TOPAS-nBio, a graphical user interface was developed. TOPAS-nBio offers full track-structure Monte Carlo simulations, integration of chemical reactions within the first millisecond, an extensive catalogue of specialized cell geometries as well as sub-cellular structures such as DNA and mitochondria, and interfaces to mechanistic models of DNA repair kinetics. We compared TOPAS-nBio simulations to measured and published data of energy deposition patterns and chemical reaction rates (G values). Our simulations agreed well within the experimental uncertainties. Additionally, we expanded the chemical reactions and species provided in Geant4-DNA and developed a new method based on independent reaction times (IRT), including a total of 72 reactions classified into 6 types between neutral and charged species. Chemical stage simulations using IRT were a factor of 145 faster than with step-by-step tracking. Finally, we applied the geometric/chemical modeling to obtain initial yields of double-strand breaks (DSBs) in DNA fibers for proton irradiations of 3 and 50 MeV and compared the effect of including chemical reactions on the number and complexity of DSB induction. Over half of the DSBs were found to include chemical reactions with approximately 5% of DSBs caused only by chemical reactions. In conclusion, the TOPAS-nBio extension to the TOPAS MC application offers access to accurate and detailed multiscale simulations, from a macroscopic description of the radiation field to microscopic description of biological outcome for selected cells. TOPAS-nBio offers detailed physics and chemistry simulations of radiobiological experiments on cells simulating the initially induced damage and links to models of DNA repair kinetics.

A New Standard DNA Damage (SDD) Data Format.

Our understanding of radiation-induced cellular damage has greatly improved over the past few decades. Despite this progress, there are still many obstacles to fully understand how radiation interacts with biologically relevant cellular components, such as DNA, to cause observable end points such as cell killing. Damage in DNA is identified as a major route of cell killing. One hurdle when modeling biological effects is the difficulty in directly comparing results generated by members of different research groups. Multiple Monte Carlo codes have been developed to simulate damage induction at the DNA scale, while at the same time various groups have developed models that describe DNA repair processes with varying levels of detail. These repair models are intrinsically linked to the damage model employed in their development, making it difficult to disentangle systematic effects in either part of the modeling chain. These modeling chains typically consist of track-structure Monte Carlo simulations of the physical interactions creating direct damages to DNA, followed by simulations of the production and initial reactions of chemical species causing so-called "indirect" damages. After the induction of DNA damage, DNA repair models combine the simulated damage patterns with biological models to determine the biological consequences of the damage. To date, the effect of the environment, such as molecular oxygen (normoxic vs. hypoxic), has been poorly considered. We propose a new standard DNA damage (SDD) data format to unify the interface between the simulation of damage induction in DNA and the biological modeling of DNA repair processes, and introduce the effect of the environment (molecular oxygen or other compounds) as a flexible parameter. Such a standard greatly facilitates inter-model comparisons, providing an ideal environment to tease out model assumptions and identify persistent, underlying mechanisms. Through inter-model comparisons, this unified standard has the potential to greatly advance our understanding of the underlying mechanisms of radiation-induced DNA damage and the resulting observable biological effects when radiation parameters and/or environmental conditions change.

Effects of heavy ions and energetic protons on normal human fibroblasts.

At the low particle fluences of radiation to which astronauts are exposed in space, "non-targeted" effects such as the bystander response may have increased significance. The radiation-induced bystander effect is the occurrence of biological responses in unirradiated cells near to or sharing medium with cells traversed by radiation. The objectives of this study were to establish the responses of AG01522 diploid human fibroblasts after exposure to several heavy ions and energetic protons, as compared to X-rays, and to obtain initial information on the bystander effect in terms of cell clonogenic survival after Fe ion irradiation. Using a clonogenic survival assay, relative biological effectiveness (RBE) values at 10% survival were 2.5, 2.3, 1.0 and 1.2 for 1 GeV/amu Fe, 1 GeV/amu Ti, 290 MeV/amu C and 1 GeV/amu protons, respectively, compared to 250 kVp X-rays. For induction of micronuclei (MN), compared to the low LET protons, Fe and Ti are very effective inducers of damage, although C ions are similar to protons. Using a transwell insert system in which irradiated and unirradiated bystander cells share medium but are not touching each other, it was found that clonogenic survival in unirradiated bystander cells was decreased when irradiated cells were exposed to Fe ions or X-rays. The magnitude of the decrease in bystander survival was similar with both radiation types, reaching a plateau of about 80% survival at doses of about 0.5 Gy or larger.

Apoptosis induced by dithiothreitol in HL-60 cells shows early activation of caspase 3 and is independent of mitochondria.

Previous studies have shown that under certain conditions some thiol-containing compounds can cause apoptosis in a number of different cell lines. Herein, we investigated the apoptotic pathways in HL-60 cells triggered by dithiothreitol (DTT), used as a model thiol compound, and tested the hypothesis that thiols cause apoptosis via production of hydrogen peroxide (H2O2) during thiol oxidation. The results show that, unlike H2O2, DTT does not induce apoptosis via a mitochondrial pathway. This is demonstrated by the absence of early cytochrome c release from mitochondria into the cytosol, the lack of mitochondrial membrane depolarization at early times, and the minor role of caspase 9 in DTT-induced apoptosis. The first caspase activity detectable in DTT-treated cells is caspase 3, which is increased significantly 1 - 2 h after the start of DTT treatment. This was shown by following the cleavage of both a natural substrate, DFF-45/ICAD, and a synthetic fluorescent substrate, z-DEVD-AFC. Cleavage of substrates of caspases 2 and 8, known as initiator caspases, does not start until 3 - 4 h after DTT exposure, well after caspase 3 has become active and at a time when apoptosis is in late stages, as shown by the occurrence of DNA fragmentation to oligonucleosomal-sized pieces. Although oxidizing DTT can produce H2O2, data presented here indicate that DTT-induced apoptosis is not mediated by production of H2O2 and occurs via a novel pathway that involves activation of caspase 3 at early stages, prior to activation of the common 'initiator' caspases 2, 8 and 9.

Quantitation of hydroxyl radicals produced by radiation and copper-linked oxidation of ascorbate by 2-deoxy-D-ribose method.

We have established controlled conditions for studying the reaction of chemically and radiolytically produced hydroxyl radical (.OH) with 2-deoxy-D-ribose (2-DR). Ascorbate (ASC) or dithiothreitol (DTT) and cuprous or cupric ions were used to generate the OH-radical. The OH-radical was detected using the classical method of measuring the amount of thiobarbituric acid reactive products (TBARP) formed by .OH-mediated 2-DR degradation, but using sensitive fluorescent detection of the TBARP production to quantify the OH-radical. All experiments were performed with adequate O(2) concentrations. The copper reaction with ASC consumes O(2) in a manner that is strongly dependent on copper concentration, and less dependent on ascorbate concentration. For an independent check of the Cu2+ catalyzed ASC oxidation kinetics, the decay of ASC absorbency at 265 nm, as well as the increase of H(2)O(2) absorbency at approximately 240 nm, were also monitored. These spectral changes agree well with the O(2) consumption data. TBARP production from 2-DR incubated with a Cu2+-ASC mixture or gamma-irradiated were also compared. gamma-Irradiation of 2-DR solutions shows a dose and 2-DR concentration dependent increase of TBARP generation. Other electron donors, such as DTT, are more complicated in their mechanism of OH-radical production. Incubation of 2-DR with Cu2+-DTT mixtures shows a delay (approximately 50 min) before OH-radical generation is detected. Our results suggest that the Cu2+-ASC reaction can be used to mimic the effects of ionizing radiation with respect to OH-radical generation. The good reproducibility and relative simplicity of the 2-DR method with fluorescence detection indicates its usefulness for the quantitation of the OH-radical generated radiolytically or chemically in carefully controlled model systems.

Effect of varying the concentration of damage restituting species in the radical repair model.

From analytical expressions derived for the radical-repair (competition) model describing the relationship between cellular radiosensitivity and oxygen concentration, "K-curve" behavior has been quantified as a function of the concentration of the species S which restitutes the radiation-induced radicals to their original molecular configuration. If these species are identified with thiols, K-curves modified by fractionally depleting [S] through calculation can be compared with experimental data where cells have their thiols depleted using various means, for example, by chemical agents or by the use of cells with decreased thiols because of genetic deficiency. Families of curves have been calculated related both to the S-depleted and the non-S-depleted hypoxic control, the latter of which is used to calculate enhancement ratios. Comparison of the model with experimental data is made.

Role of glutathione in the aerobic radiation response.

We will review the relationships between glutathione (GSH), protein thiols, and cellular responses to radiation, peroxides, and peroxide-producing drugs. Our primary interest involves the behavior of sulfhydryls as electron and hydrogen carriers, and their capacity to protect various target molecules against radiation and peroxidative damage. We used reagents such as L-buthionine sulfoximine (LBSO), alone and in combination with N-ethyl maleimide (NEM), diamide, and dimethylfumarate, to decrease GSH so that it could no longer participate in the electron transfer reactions. Our results indicate that aerobic sensitization produced by GSH depletion can be further enhanced if electron-accepting agents, such as tertiary butyl hydroperoxide (t-BOOH), are present during irradiation. Hydroperoxide is a substrate for glutathione peroxidase and diverts electrons and hydrogen away from target molecules during its reduction. Sensitivity to radiation seems to be due to the inhibition of the mitochondria's capacity to reduce hydroperoxide. We will also report the mitochondria's ability to reduce the oxygen radicals produced by radiation and drugs. Data also indicate that t-BOOH oxidizes protein thiols which are enzymatically involved in repair of DNA damage.

Interactions of radioprotectors and oxygen in cultured mammalian cells. I. Dithiothreitol effects on radiation-induced cell killing.

Radioprotection in vitro by sulfhydryl (SH)-containing compounds is usually greater in aerated than in hypoxic cells. This observation has been cited recently as one of the reasons for the relatively greater effectiveness of radioprotectors such as WR-2721 in normal tissues compared to tumor cells. It is demonstrated herein, however, that hypoxic V79 cells irradiated in vitro under carefully controlled conditions are protected to a greater extent by low concentrations (1-2 mM) of the SH compound dithiothreitol (DTT) than are aerated cells. The reverse, more general phenomenon is seen at high concentrations of DTT (greater than 2 mM). This complex SH concentration and oxygenation dependence results in an increase in the oxygen enhancement ratio (OER) at low concentrations of DTT relative to the OER in the absence of DDT, followed by a decrease in OER at concentrations greater than 2 mM DTT. The possible radiation chemical basis for this finding and its importance to the clinical use of SH-containing radioprotectors are discussed.

Mechanisms for the oxygen radical-mediated toxicity of various thiol-containing compounds in cultured mammalian cells.

When Chinese hamster V79 cells are exposed to various thiol compounds in phosphate-buffered saline (PBS), some compounds cause toxicity (loss of colony formation), although the dependence on drug concentration and the magnitude of the cell killing vary between the different thiols. For example: dithiothreitol (DTT) and WR-1065 cause a biphasic toxicity whereby cell killing occurs at about 0.2 to 1.0 mM thiol, but is not seen at higher or lower drug concentrations; N-acetylcysteine (NAC) is toxic only at concentrations > or = 2 mM and shows no biphasic pattern; and glutathione (GSH) and penicillamine are only minimally toxic at all concentrations. The effect of the addition of 1 microM Cu2+ to the thiol also depends on the particular thiol: e.g., Cu2+ increases cell killing in the biphasic pattern with WR-1065; it increases the toxicity of NAC only at high thiol concentrations; and it elicits a slight toxicity in the biphasic pattern by GSH and penicillamine. In all cases tested, if the thiol is toxic, the cell killing can be decreased or prevented by addition of catalase, consistent with the hypothesis that the toxicity is mediated through H2O2 produced during the thiol oxidation. However, when the oxidation rates of the various thiols in PBS without and with Cu2+ were measured, the data did not show a simple correlation between the toxicity of the various thiols and their oxidation rates. The rate of the reaction of the various thiols with H2O2 was also determined and showed a better, but still not good, correlation with toxicity. However, cell killing by the various thiols correlated better with the ratio between the half-lives for thiol oxidation and reaction of thiol with H2O2 than with either reaction rate alone. This suggests that the toxicity pattern and magnitude of cell killing in V79 cells by various thiols depend on the interplay between the rate of thiol oxidation and the rate of reaction between the thiol and the H2O2 produced in the thiol oxidation.

DNA damage measured using the alkaline elution assay depends on time between subculturing of cells and irradiation.

In experiments utilizing the alkaline filter elution assay for radiation-induced DNA damage we observed an unexpected dependence of hypoxic dose-response curves on the length of time V79 cells were in exponential growth between subculturing and irradiation. Dose-response curves for DNA from cells irradiated in air were identical regardless of whether the exponential-phase cells had been subcultured 24 or 48 h prior to irradiation, but cells irradiated in hypoxia 24 h after subculture displayed a dose-response curve for DNA damage which was two times steeper than that obtained for cells irradiated in hypoxia 48 h after subculture. Possible mechanisms for this effect are discussed.

Role of copper in the oxygen radical-mediated toxicity of the thiol-containing radioprotector dithiothreitol in mammalian cells.

The thiol radioprotector dithiothreitol (DTT) causes biphasic toxicity in V79 cells: exposure to DTT causes loss of clonogenic cell survival at intermediate concentrations of the drug, but is not toxic at lower (< 0.05 mM) or higher (> 2.0 mM) concentrations. Toxicity depends on the medium in which cells are exposed to DTT. Cell killing is less when cells are exposed to DTT in phosphate-buffered saline (PBS) than when in complete, serum-containing medium, and there is no cell killing at any drug concentration when cells are in serum-free medium. When cells are exposed to DTT in PBS containing 10% fetal calf serum (FCS) or 10% dialyzed FCS, cell killing is increased compared to the response in PBS alone, suggesting that some component(s) of serum is involved in DTT toxicity. Addition of micromolar quantities of copper as either free Cu2+ or ceruloplasmin to serum-free medium increases the toxicity of DTT, but addition of free iron or transferrin has no effect. H2O2 is produced during DTT oxidation and appears to be involved in the toxicity of DTT, because toxicity can be prevented by catalase. H2O2 is also toxic to V79 cells in a medium-dependent fashion, but the toxicity is not influenced by addition of copper, ceruloplasmin, iron or transferrin. The rate of DTT oxidation is also medium-dependent and is increased by copper or ceruloplasmin, but not by iron or transferrin. The data are consistent with the hypothesis that the toxicity of DTT is caused by the copper-catalyzed oxidation of DTT, forming H2O2 which, in turn, produces .OH, the ultimate toxic agent, via the Fenton reaction.

Toxicity of the sulfhydryl-containing radioprotector dithiothreitol.

The toxicity of the sulfhydryl-containing radioprotective agent dithiothreitol (DTT) has been studied using Chinese hamster V79 cells growing in monolayer in minimal essential medium containing 10% fetal calf serum. DTT at low concentrations (between 0.4 and 1.0 mM) caused cell killing, but higher concentrations (above 2 mM) or lower concentrations (0.1 mM) did not. This DTT-induced toxicity was prevented by catalase, glutathione, the use of serum-free medium, or lowering incubation temperature; was slightly decreased by dimethyl sulfoxide; and was enhanced by some metal chelators but prevented by desferal, an iron chelator. Experiments involving simultaneous exposure of cells to DTT and H2O2 showed that low concentrations of DTT enhanced H2O2-induced toxicity, but high concentrations of DTT prevented the H2O2 toxicity. These results are consistent with the proposal that toxicity results from autoxidation of DTT to produce H2O2, which in turn reacts via the metal-catalyzed Fenton reaction to produce the ultimate toxin, .OH radicals, although chemical studies show that rates of autoxidation of various sulfhydryl compounds do not correlate with the observed toxicity.

Coumarin-3-carboxylic acid as a detector for hydroxyl radicals generated chemically and by gamma radiation.

Coumarin-3-carboxylic acid (3-CCA) was used as a detector for hydroxyl radicals (.OH) in aqueous solution. The .OH was generated by gamma irradiation or chemically by the Cu2+-mediated oxidation of ascorbic acid (ASC). The excitation and emission spectra of 3-CCA, hydroxylated either chemically or by gamma irradiation, were nearly identical to those of an authentic 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). The pH-titration curves for the fluorescence at 450 nm (excitation at 395 nm) of 3-CCA, hydroxylated either chemically or by gamma radiation, were also identical to those of authentic 7-OHCCA (pK = 7.4). Time-resolved measurements of the fluorescence decays of radiation- or chemically hydroxylated 3-CCA, as well as those of 7-OHCCA, indicate a monoexponential fit. The fluorescence lifetime for the product of 3-CCA hydroxylation was identical to that of 7-OHCCA (approximately 4 ns). These data, together with analysis of end products by high-performance liquid chromatography, show that the major fluorescent product formed by radiation-induced or chemical hydroxylation of 3-CCA is 7-OHCCA. Fluorescence detection of 3-CCA hydroxylation allows real-time measurement of the kinetics of .OH generation. The kinetics of 3-CCA hydroxylation by gamma radiation is linear, although the kinetics of 3-CCA hydroxylation by the Cu2+-ASC reaction shows a sigmoid shape. The initial (slow) step of 3-CCA hydroxylation is sensitive to Cu2+, but the steeper (fast) step is sensitive to ASC. Analysis of the kinetics of 3-CCA hydroxylation shows a diffusion-controlled reaction with a rate constant 5.0 +/- 1.0 x 10(9) M(-1) s(-1). The scavenging of .OH by 3-CCA was approximately 14% for chemical generation with Cu2+-ASC and approximately 50% for gamma-radiation-produced .OH. The yield of 7-OHCCA under the same radiation conditions was approximately 4.4% and increased linearly with radiation dose. The 3-CCA method of detection of .OH is quantitative, sensitive, specific and therefore accurate. It has an excellent potential for use in biological systems.

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate.

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.

Interactions of radioprotectors and oxygen in cultured mammalian cells. II. Effects of dithiothreitol on radiation-induced DNA damage and comparison with cell survival.

The effects of the sulfhydryl-containing compound dithiothreitol (DTT) on radiation-induced DNA damage have been studied using two different assays: DNA unwinding hydroxyapatite chromatography and alkaline filter elution. DNA damage as measured by both assays for cells irradiated in air shows drug concentration-dependent radioprotection reaching high levels (dose reduction factor, DRF = 3) at high DTT concentrations. The pattern and degree of protection against DNA damage are the same as shown previously for cell survival. However, when cells are irradiated in hypoxia, DNA damage as measured by the unwinding technique is decreased less by low DTT concentrations than is survival, but DNA damage is decreased to a much greater extent (DRF = 3) at high concentrations of DTT (compared to DRF = 1.5 for cell survival). DNA damage as measured by the alkaline elution assay after hypoxic irradiation is decreased to a much greater extent at all concentrations of DTT with DRF = 1.6 at 1 mM and increasing to DRF = 4.5 at high levels of DTT. These results are discussed in terms of the different types of DNA damage produced in cells irradiated in air versus hypoxia and the differences in types of damage measured by the two different DNA assays and cell survival.

Role of the pentose cycle in oxygen radical-mediated toxicity of the thiol-containing radioprotector dithiothreitol in mammalian cells.

We have shown previously that the thiol-containing radioprotector dithiothreitol (DTT) kills V79 cells in a manner that is dependent on both the concentration of DTT and the medium. The results are consistent with the hypothesis that DTT toxicity is caused by the copper-catalyzed oxidation of DTT, forming H2O2, which in turn produces .OH, the ultimate toxic species, via the metal-catalyzed Fenton reaction. Because it is known that the pentose cycle plays a role in the ability of cells to deal with oxidative stress, the hypothesis that the pentose cycle is involved in the response of cells to DTT is tested in this paper. The results show that toxicity of both DTT and H2O2 in V79 cells is greater in cells exposed to the drugs in medium lacking glucose than in cells in medium containing glucose. Addition of glucose to medium or buffer lacking it decreases DTT- and H2O2-induced cell killing. Studies using cells deficient in glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose cycle, show that cells of the mutant cell lines (E16 and E48) are more sensitive to cell killing by both DTT and H2O2 than are the parental CHO K1D cells when exposed to the drugs in medium containing glucose. However, toxicity does not differ significantly among the three cell lines when they are exposed to DTT or H2O2 in phosphate-buffered saline that lacks glucose. Measurements of pentose cycle activity show that the pentose cycle in K1D cells is stimulated by DTT, with the pattern and drug concentration dependence of the stimulation being similar to that for cell killing. However, the pentose cycle is not stimulated by DTT in G6PD-deficient cell lines. The data are consistent with the hypothesis that the pentose cycle is one of the cellular pathways that mediates the oxidative stress imposed by DTT or H2O2.

Pulse radiolysis studies of the interactions of the sulfhydryl compound dithiothreitol and sugars.

Pulse radiolysis studies of the hydrogen atom transfer ("repair") reaction from the sulfhydryl-containing (RSH) compound dithiothreitol (DTT) to the DNA sugar deoxyribose and to several related sugars have been undertaken. The H transfer reaction is measured by monitoring the transient absorbance of the radical-anion RSSR-. The H atom transfer reactions for some sugars were fitted by a single time exponential function, but other sugars exhibited both a fast and a slow component (approximately 10-fold difference in rates) to the reaction. The reaction rates for the slow stage of the reaction between DTT and the sugars ranged from 0.5 X 10(7) dm3 mole-1 sec-1 for ribose-5-phosphate to 9 X 10(7) dm3 mole-1 sec-1 for 2-deoxyglucose. The maximum extent of total repair varied from 60% for ribose-5-phosphate to 100% for 2-deoxyglucose. The rate of repair, the extent of repair, and the appearance of more than one component of repair seem to depend on several factors: The occurrence of radical-radical reactions in the system is indicated by the demonstration of a dose dependence of the reaction kinetics, and this affects the observed rate of formation of RSSR-. Sugars with a deoxy group on the 2-carbon atom seem to have enhanced rates and extents of repair and to exhibit both fast and slow components to the reaction. The presence of a phosphate group on the sugar causes a decrease in the rate and extent of repair. The biological relevance of the reactions studied herein is discussed and the rates obtained are compared with rates for repair of damage in certain radiobiological systems.

Role of Fenton chemistry in thiol-induced toxicity and apoptosis.

Under certain conditions, many radioprotective thiols can be toxic, causing loss of colony-forming ability in cultured mammalian cells in a biphasic fashion whereby the thiols are not toxic at high or low concentrations of the drug, but cause decreased clonogenicity at intermediate (0.2-1.0 mM) drug levels. This symposium paper summarizes our studies using dithiothreitol (DTT) as a model thiol to demonstrate the role of Fenton chemistry in thiol toxicity. The toxicity of DTT in V79 cells has several characteristics: it is dependent on the medium used during exposure of cells to the drug; the toxicity is decreased or prevented by addition of catalase exogenously, but superoxide dismutase has no effect; the toxicity is increased by addition of copper, either free or derived from ceruloplasmin in serum; and the toxicity can be modified intracellularly by altering glucose availability or pentose cycle activity. Thus the data are consistent with a mechanism whereby DTT oxidation produces H2O2 in a reaction catalyzed by metals, predominantly copper, followed by reaction of H2O2 in a metal-catalyzed Fenton reaction to produce the ultimate toxic species, .OH. Studies comparing 12 thiols have shown that the magnitude of cell killing and pattern of dependence on thiol concentration vary among the different agents, with the toxicity depending on the interplay between the rates of two reactions: thiol oxidation and the reaction between the thiol and the H2O2 produced during the thiol oxidation. The addition of other metals, e.g. Zn2+, and metal chelators, e.g. EDTA, can also alter DTT toxicity by altering the rates of thiol oxidation or the Fenton reaction. Recent studies have shown that in certain cell lines thiols can also cause apoptosis in a biphasic pattern, with little apoptosis at low or high drug concentrations but greatly increased apoptosis levels at intermediate (approximately 3 mM) thiol concentrations. There appears to be a good correlation between those thiols that cause loss of clonogenicity and those that induce apoptosis, suggesting similar mechanisms may be involved in both end points. However, thiol-induced apoptosis is not prevented by addition of exogenous catalase. These observations are discussed in relation to the possible role of Fenton chemistry in induction of apoptosis by thiols.

Role of guanosine triphosphate in ferric ion-linked Fenton chemistry.

We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.