Biological consequences of radiation-induced DNA damage: relevance to radiotherapy.

DNA damage of exposed tumour tissue leading to cell death is one of the detrimental effects of ionising radiation that is exploited, with beneficial consequences, for radiotherapy. The pattern of the discrete energy depositions during passage of the ionising track of radiation defines the spatial distribution of lesions induced in DNA with a fraction of the DNA damage sites containing clusters of lesions, formed over a few nanometres, against a background of endogenously induced individual lesions. These clustered DNA damage sites, which may be considered as a signature of ionising radiation, underlie the deleterious biological consequences of ionising radiation. The concepts developed rely in part on the fact that ionising radiation creates significant levels of clustered DNA damage, including complex double-strand breaks (DSB), to kill tumour cells as clustered damage sites are difficult to repair. This reduced repairability of clustered DNA damage using specific repair pathways is exploitable in radiotherapy for the treatment of cancer. We discuss some potential strategies to enhance radiosensitivity by targeting the repair pathways of radiation-induced clustered damage and complex DNA DSB, through inhibition of specific proteins that are not required in the repair pathways for endogenous damage. The variety and severity of DNA damage from ionising radiation is also influenced by the tumour microenvironment, being especially sensitive to the oxygen status of the cells. For instance, nitric oxide is known to influence the types of damage induced by radiation under hypoxic conditions. A potential strategy based on bioreductive activation of pro-drugs to release nitric oxide is discussed as an approach to deliver nitric oxide to hypoxic tumours during radiotherapy. The ultimate aim of this review is to stimulate thinking on how knowledge of the complexity of radiation-induced DNA damage may contribute to the development of adjuncts to radiotherapy.

Measurement of plasma 5-hydroxyindoleacetic acid as a possible clinical surrogate marker for the action of antivascular agents.

BACKGROUND: Serotonin (5HT), a naturally occurring vasoactive substance, is released from platelets into plasma under various pathological conditions. Recently, anticancer drugs that act by selectively disrupting tumour blood flow have been found to increase plasma 5HT concentrations in mice. Two such antivascular agents, flavone acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have completed Phase I clinical trial and raise the important question of whether suitable surrogate markers for antivascular effects can be identified. METHODS: 5HT is unstable to storage, precluding routine clinical assay, but the 5HT metabolite, 5-hydroxyindoleacetic acid (5HIAA) accumulates in plasma following 5HT release and is a more suitable marker because of its greater stability. We have developed an automated procedure for the assay of the low concentrations of 5HIAA found in humans by combining solid-phase extraction with high-performance liquid chromatography (HPLC). RESULTS: Efficient separation of 5HIAA from possible interfering substances in human plasma, including a variety of pharmaceutical agents, was achieved on C18 columns using cetyltrimethylammonium bromide (CETAB) as an organic modifier. Adequate precision, accuracy and sensitivity were achieved by electrochemical detection (ECD) at +400 mV. Analysis of plasma from two patients treated with DMXAA in a Phase I trial demonstrated DMXAA-induced elevation of plasma 5HIAA with a time course similar to that previously described in mice. CONCLUSIONS: Measurement of changes in plasma 5HIAA provides a new approach to the monitoring of therapies with an antivascular effect. The assay is sensitive to dietary sources of 5HT, which should be minimised.

Influence of plasma glutathione levels on radiation mucositis.

PURPOSE: To test the hypothesis that there is a link between plasma glutathione (GSH) or other antioxidants (uric acid, ascorbate) and the severity of radiation mucositis following radiation treatment of tumors of the head and neck. PATIENTS AND METHODS: Patients with carcinomas of the head-and-neck region were treated with the continuous hyperfractionated accelerated radiotherapy (CHART) regimen (54 Gy in 36 fractions over 12 days). Samples of blood plasma were analyzed for GSH, cysteine, urate, and ascorbate by high-pressure liquid chromatography. Patients were graded for dysphagia and requirement for analgesics. The areas under the curves of scores over 2-6 weeks following treatment were computed, and Spearman's rank-correlation coefficient was used to test for an association between plasma GSH levels (or those of other antioxidants) and mucositis. RESULTS: The pretreatment plasma GSH level in 18 patients scored in the study was 1.0 +/- 0.7 M. Analysis of these and the dysphagia scores produced a correlation coefficient of 0.22 (confidence interval -0.28, 0.61; p = 0.39). No correlation was seen between mucositis severity and other measures of plasma antioxidants: cysteine (7.6 +/- 1.7 M), cysteine + GSH (8.6 +/- 1.9 M), uric acid (317 +/- 86 M), ascorbate (29 +/- 20 M), or whole-blood GSH concentrations (1,010 +/- 239 M). CONCLUSION: The measurements of approximately micromolar levels of plasma GSH, or about 10 M cysteine + GSH (almost all of the total nonprotein thiols), are consistent with most other published data for either healthy adults or cancer patients; however, the values reported in an earlier study, suggesting a link between GSH and mucositis, are much higher. The hypothesis of a possible link between radiation mucositis and plasma-free (nonprotein) thiols was not supported.

Oxidative activation of indole-3-acetic acids to cytotoxic species- a potential new role for plant auxins in cancer therapy.

Indole-3-acetic acid (IAA) and some derivatives can be oxidised by horseradish peroxidase (HRP) to cytotoxic species. Upon treatment with IAA/HRP, liposomes undergo lipid peroxidation, strand breaks and adducts are formed in supercoiled plasmid DNA, and mammalian cells in culture lose colony-forming ability. IAA is only toxic after oxidative decarboxylation; no effects are seen when IAA or HRP is incubated independently in these systems at equivalent concentrations. Toxicity is similar in both hamster fibroblasts and some human tumour cells. The effect of IAA/HRP is thought to be due in part to the formation of 3-methylene-2-oxindole, which may conjugate with DNA bases and protein thiols. Our hypothesis is that IAA/HRP could be used as the basis for targeted cancer therapy involving antibody-, polymer-, or gene-directed approaches. HRP can thus be targeted to a tumour allowing non-toxic IAA delivered systemically to be activated only in the tumour. Exposure to newly synthesised analogues of IAA shows a range of four orders of magnitude difference in cellular toxicity but no structure-activity relationships are apparent, in contrast to well-defined redox dependencies of oxidation by HRP intermediates or rates of decarboxylation of radical-cation intermediates.

Horseradish peroxidase-mediated gene therapy: choice of prodrugs in oxic and anoxic tumor conditions.

We have previously proposed the plant enzyme horseradish peroxidase (HRP) and the plant hormone indole-3-acetic acid (IAA) as an enzyme/prodrug combination for cancer gene therapy. In the current study, we evaluated the potential of HRP/IAA for gene-directed enzyme/prodrug therapy in three human tumor cell lines (T24 bladder carcinoma, MCF-7 breast adenocarcinoma, and FaDu nasopharyngeal squamous carcinoma) and one endothelial cell line (HMEC-1). The action of 10 IAA analogues in combination with HRP was studied in vitro in normoxic conditions as well as in the extreme tumor conditions of anoxia. Compounds characterized by prompt normoxic or anoxic cytotoxic activation and high HRP transfectant killing or selectivity were identified. Some variations were observed in the response of cells of different origin, with IAA, 1-Me-IAA, and 5-Br-IAA representing the most promising candidates for HRP gene therapy. In particular, 5-Br-IAA showed a very prompt and selective activation in anoxia. A strong bystander effect was produced by activated IAA and analogues because 70-90% cell kill was obtained when only 5% of the cells expressed the HRP enzyme. These results indicate that HRP/IAA represents an effective system for enzyme/prodrug-based anticancer approaches, and further improvements could be achieved by the use of novel IAA derivatives.

Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid.

This paper demonstrates the potential for utilizing the plant enzyme, horseradish peroxidase (HRP), in a gene-directed enzyme prodrug therapy context. Human T24 bladder carcinoma cells transfected with a mammalian expression vector containing the HRP cDNA were selectively sensitized to the nontoxic plant hormone, indole-3-acetic acid (IAA). The HRP/IAA-induced cell kill was effective in normoxic and anoxic conditions. The activated drug is a long-lived species able to cross cell membranes, and cell contact appears not to be required for a bystander effect to take place. These preliminary results suggest that the delivery of the HRP gene to human tumors followed by IAA treatment may provide a novel cancer gene-directed enzyme prodrug therapy approach, with potential to target hypoxic cells.

Effect of local radiotherapy for bone pain on urinary markers of osteoclast activity.

Urinary markers of bone resorption, pyridinoline and deoxypyridinoline were measured before and at 4 weeks after radiotherapy for metastatic bone pain. An association was shown between relief of metastatic skeletal pain by radiotherapy and low marker concentrations before and after treatment, lending support to the hypothesis that relief of metastatic bone pain by radiotherapy relates to an effect on bone, rather than tumour physiology.

Peroxidase-catalyzed effects of indole-3-acetic acid and analogues on lipid membranes, DNA, and mammalian cells in vitro.

This study aimed to explore the mechanisms and molecular parameters which control the cytotoxicity of derivatives of indole-3-acetic acid (IAA) when oxidatively activated by horseradish peroxidase (HRP). Lipid peroxidation was measured in liposomes, damage to supercoiled plasmid DNA assessed by gel electrophoresis, free radical intermediates detected by EPR following spin trapping, binding of IAA-derived products demonstrated by 3H labelling, stable products measured by HPLC, and cytotoxicity in hamster fibroblasts measured by clonogenic survival. IAA, and nine analogues more easily oxidized by HRP, caused lipid peroxidation in liposomes, but not detectably in membranes of hamster fibroblasts, and were cytotoxic after HRP activation to varying degrees. Cytotoxicity was not correlated with activation rate. The hydrophilic vitamin E analogue, Trolox, inhibited cytotoxicity, whereas loading fibroblasts with vitamin E was ineffective, consistent with an oxidative mechanism in which radical precursors to damage are intercepted by Trolox in the aqueous phase. However, two known oxidation products were nontoxic (the 3-carbinol and 3-aldehyde, both probably produced from 3-CH2OO* peroxyl radicals via the 3-CH*2 [skatolyl] radical following decarboxylation of the radical cation). The skatolyl radical from IAA was shown by EPR with spin trapping to react with DNA; electrophoresis showed binding to occur. Treatment of hamster fibroblasts with 5-3H-IAA/HRP resulted in intracellular bound 3H. Together with earlier results, the new data point to unknown electrophilic oxidation products, reactive towards intracellular targets, being involved in cytotoxicity of the IAA/HRP combination, rather than direct attack of free radicals, excited states, or membrane lipid peroxidation.

Toward targeted "oxidation therapy" of cancer: peroxidase-catalysed cytotoxicity of indole-3-acetic acids.

PURPOSE: The study aimed to identify suitable prodrugs that could be used to test the hypothesis that peroxidase activity in cells, either endogenous or enhanced by immunological targeting, can activate prodrugs to cytotoxins. We hypothesized that prototype prodrugs based on derivatives of indole-3-acetic acid (IAA), when activated by peroxidase enzymes (e.g., from horseradish, HRP) should produce peroxyl radicals, with deleterious biological consequences. METHODS AND MATERIALS: V79 hamster cells were incubated with IAA or derivatives +/- HRP and cytotoxicity assessed by a clonogenic assay. To assess the toxicity of stable oxidation products, prodrugs were also oxidized by HRP without cells, and the products then added to cells. RESULTS: The combination of prodrug and enzyme resulted in cytotoxicity, but neither indole nor enzyme in isolation was toxic under the conditions used. Although lipid peroxidation was stimulated in liposomes by the prodrug/enzyme treatment, it could not be measured in mammalian cells. Adding oxidized prodrugs to cells resulted in cytotoxicity. CONCLUSIONS: Although the hypothesis that prodrugs of this type could enhance oxidative stress via lipid peroxidation was not established, the results nonetheless demonstrated oxidatively-activated cytotoxicity via indole acetic acid prodrugs, and suggested these as a new type of substrate for antibody-directed enzyme-prodrug therapy (ADEPT). The hypothesized free-radical fragmentation intermediates were demonstrated, but lipid peroxidation associated with peroxyl radical formation was unlikely to be the major route to cytotoxicity.

Interpretation of the reactivity of peroxidase compounds I and II with phenols by the Marcus equation.

The catalytic cycle of heme peroxidases involves two reactive states, compound I and compound II. Although their reduction potentials at pH 7 are similar, compound I is in general more reactive towards organic substrates than compound II. The different reactivities have until now remained unexplained. In this study, the reactions of compounds I and II of peroxidase from horseradish with phenols were analyzed using the Marcus equation of electron-transfer. Both reactions exhibit similar reorganization energies, and the different reactivities of the two enzyme states can be ascribed to a higher apparent rate of activationless electron-transfer in the compound I reactions. This can be attributed to the shorter electron-tunneling distance on electron-transfer to the porphyrin radical cation in compound I, compared to electron-transfer to the iron ion in compound II.

Factors controlling the substrate specificity of peroxidases: kinetics and thermodynamics of the reaction of horseradish peroxidase compound I with phenols and indole-3-acetic acids.

The rates of oxidation of reducing substrates by heme peroxidases have previously been thought to be controlled only by their ease of oxidation. In the present study, we have compared the kinetics and thermodynamics of the oxidation of indole-3-acetic acid and derivatives and of phenols by horseradish peroxidase. Different dependencies of the reaction rates on the thermodynamic driving force reveal substrate specificity controlled by the enzyme-substrate complexes dissociation constants (Michaelis-Menten constants) and by the reorganization energies of electron-transfer within those complexes.

Rates of reaction of indoleacetic acids with horseradish peroxidase compound I and their dependence on the redox potentials.

The rates of reaction of seven indole-3-acetic acid derivatives with horseradish peroxidase compound 1 at pH 5 were measured by stopped flow, and the reduction potentials and pKa of their radical cations were determined by pulse radiolysis. Reasonable correlation of these properties with Hammett substituent parameters was found, but not with Brown-Okamoto (theta +) parameters. The rates of reaction with compound I correlate well with the reduction potentials under the same conditions, with rates of reaction that increase by ca. 2.5 orders of magnitude with a 100 mV decrease in the reduction potential. This relationship is in agreement with that previously estimated for the reaction of compound I with phenols and anilines, suggesting that the rate of reaction depends solely on the reduction potential of the substrate radical, even for compounds of dissimilar structure.

Enhancement of peroxidase-induced lipid peroxidation by indole-3-acetic acid: effect of antioxidants.

Indole-3-acetic acid (IAA) enhanced the peroxidase-induced lipid peroxidation in phosphatidylcholine liposomes, as measured by loss of fluorescence of cis-parinaric acid. α-Tocopherol or ß-carotene in the lipid phase or ascorbate or Trolox in the aqueous phase inhibited the loss of fluorescence induced by the peroxidase + IAA system, but glutathione had only a small inhibitory effect. The peroxyl radical formed by one-electron oxidation of IAA, followed by decarboxylation and reaction with oxygen, is suggested to act as the initiator of lipid peroxidation. The protection by ascorbate or Trolox is explained by the reactivity of these compounds with the IAA indolyl radical, as shown by pulse radiolysis experiments, whereas the weak effect of glutathione agrees with its low reactivity towards the IAA-derived peroxyl radical and its precursors.

Enhancement of lipid peroxidation by indole-3-acetic acid and derivatives: substituent effects.

The peroxidation of liposomes by a haem peroxidase and hydrogen peroxide in the presence of indole-3-acetic acid and derivatives was investigated. It was found that these compounds can accelerate the lipid peroxidation up to 65 fold and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. The peroxyl radicals are formed by peroxidase-catalyzed oxidation of the enhancers to radical cations which undergo cleavage of the carbon-carbon bond on the side-chain to yield CO2 and carbon-centred radicals that rapidly add oxygen. In competition with decarboxylation, the radical cations deprotonate reversibly from the N1 position. Rates of decarboxylation, pka values and rate of reaction with the peroxidase compound I indicate consistent substituent effects which, however, can not be quantitatively related to the usual Hammett or Brown parameters. Assuming that the rate of decarboxylation of the radical cations taken is a measure of the electron density of the molecule (or radical), it is found that the efficiency of these compounds as enhancers of lipid peroxidation increases with increasing electron density, suggesting that, at least in the model system, the oxidation of the substrates is the limiting step in causing lipid peroxidation.

Kinetics and mechanisms of hypochlorous acid reactions.

Hypochlorous acid (HOCl) is a strong oxidant formed in neutrophils by the myeloperoxidase-catalyzed oxidation of chloride. Using stopped-flow with spectrophotometric detection, HOCl was found to react very rapidly with glutathione and ascorbate and less rapidly with taurine. No evidence could be found for the formation of reactive free-radical intermediates in these reactions, in support of an electrophilic mechanism. In contrast, the reaction with iron(II) aquo or citrate complexes (k approximately 10(4) dm3 mol-1 s-1 in acidic solution) yielded reactive intermediates distinguishable from hydroxyl radicals. The reaction between HOCl and ferrous ions, which is analogous to but faster than the Fenton reaction, is a potential source of free radicals in activated neutrophils.

Free-radical repair by a novel perthiol: reversible hydrogen transfer and perthiyl radical formation.

2-(3-Aminopropyl-amino) ethaneperthiol (RSSH, the perthiol analogue of the thiol radioprotector, WR-1065) reacts with the alpha-hydroxy alkyl radical (CH3)2C.OH by donating a hydrogen atom as indicated by the characterization of perthiyl radicals (RSS.; lambda max approximately 374 nm, epsilon 374 approximately 1680 +/- 20 dm3 mol-1 cm-1) by pulse radiolysis. The perthiyl radical abstracts a hydrogen from the alcohol to establish a reversible hydrogen-transfer equilibrium. This equilibrium lies predominantly on the side of radical repair since the rate constants for the forward and reverse reactions at pH 4 are: kappa(RSSH+(CH3)2C.OH) = (2.4 +/- 0.1) x 10(9) dm3 mol-1 s-1 and kappa(RSS.+(CH3)2CHOH) = (3.8 +/- 0.3) x 10(3) dm3 mol-1 s-1 respectively. The pKa (RSSH<-->RSS(-)+H+) = 6.2 +/- 0.1 was determined from the pH dependence of the rate of perthiol repair. Identical experiments have been performed with WR-1065 allowing a direct comparison of free-radical repair reactivity to be made with the parthiol analogue. At pH approximately 7.4 the reactivities of the thiol and perthiol were similar, both repairing the alcohol radical with a rate constant of approximately (2.4 +/- 0.1) x 10(8) dm3 mol-1 s-1. However, at pH 5 whilst the hydrogen-donation rate of the thiol was 15-20% higher than at pH 7.4, the perthiol reactivity was over an order of magnitude higher. The thermodynamic driving force for the observed enhanced free-radical repair reactivity of RSSH compared to RSH is attributed to the resonance stabilization energy of 8.8 kJ mol-1 within the RSS. radical. These results indicate a possible application of RSSH/RSS- as DNA-targeted antioxidants or chemoprotectors.