Smoking in help-seeking veterans with PTSD returning from Afghanistan and Iraq.

Past research has shown that veterans and individuals with posttraumatic stress disorder (PTSD) have increased rates of smoking. However, the rates of smoking in younger help-seeking veterans returning from Afghanistan and Iraq, and possible correlates of smoking among this population are unknown. In this study, we evaluated the rate of lifetime and current smoking among a sample of 90 returning male veterans diagnosed with PTSD. Fifty-nine percent reported a lifetime history of smoking including 32% that were current smokers. Current smokers were significantly younger than non-smokers. Current smokers (mean age=31) reported a mean age of smoking onset as 15.86 with a pack year history of 8.89. These smokers reported on average five previous quit attempts. According to a stages of change model, one-half of the smokers were in the contemplation phase of stopping smoking (50%), 29% were in the pre-contemplation phase and 21% were in the preparation phase. The results are placed in the context of non-psychiatric and psychiatric smokers.

Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues.

The potential for tumor vascular-targeting by using the tubulin destabilizing agent disodium combretastatin A-4 3-0-phosphate (CA-4-P) was assessed in a rat system. This approach aims to shut down the established tumor vasculature, leading to the development of extensive tumor cell necrosis. The early vascular effects of CA-4-P were assessed in the s.c. implanted P22 carcinosarcoma and in a range of normal tissues. Blood flow was measured by the uptake of radiolabeled iodoantipyrine, and quantitative autoradiography was used to measure spatial heterogeneity of blood flow in tumor sections. CA-4-P (100 mg/kg i.p.) caused a significant increase in mean arterial blood pressure at 1 and 6 h after treatment and a very large decrease in tumor blood flow, which-by 6 h-was reduced approximately 100-fold. The spleen was the most affected normal tissue with a 7-fold reduction in blood flow at 6 h. Calculations of vascular resistance revealed some vascular changes in the heart and kidney for which there were no significant changes in blood flow. Quantitative autoradiography showed that CA-4-P increased the spatial heterogeneity in tumor blood flow. The drug affected peripheral tumor regions less than central regions. Administration of CA-4-P (30 mg/kg) in the presence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, potentiated the effect of CA-4-P in tumor tissue. The combination increased tumor vascular resistance 300-fold compared with less than 7-fold for any of the normal tissues. This shows that tissue production of nitric oxide protects against the damaging vascular effects of CA-4-P. Significant changes in tumor vascular resistance could also be obtained in isolated tumor perfusions using a cell-free perfusate, although the changes were much less than those observed in vivo. This shows that the action of CA-4-P includes mechanisms other than those involving red cell viscosity, intravascular coagulation, and neutrophil adhesion. The uptake of CA-4-P and combretastatin A-4 (CA-4) was more efficient in tumor than in skeletal muscle tissue and dephosphorylation of CA-4-P to CA-4 was faster in the former. These results are promising for the use of CA-4-P as a tumor vascular-targeting agent.

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.

Determination of combretastatin A-4 and its phosphate ester pro-drug in plasma by high-performance liquid chromatography.

High-performance liquid chromatography with both absorbance and fluorescence detection has been applied to the determination of the potential anti-tumour agent combretastatin A-4 and its phosphate ester in murine and human plasma. The presence of different interfering peaks in the two species makes absorbance detection at 295 nm the method of choice for the mouse, and fluorescence detection (295 nm/390 nm) for human plasma. The calibration was linear over the range studied (0.01-50 microM for combretastatin A-4, 0.02-200 microM for combretastatin A-4 phosphate), with quantitation limits of 0.05 microM for both drugs in the mouse, and 0.05 microM and 0.0125 microM for the phosphate ester and free drug, respectively, in human plasma. The method should be useful for pharmacokinetic studies in the forthcoming Phase I clinical trial of combretastatin A-4 phosphate.

Pharmacokinetics of nicotinamide in cancer patients treated with accelerated radiotherapy: the experience of the Co-operative Group of Radiotherapy of the European Organization for Research and Treatment of Cancer.

BACKGROUND: The EORTC has initiated studies to combine nicotinamide with carbogen in accelerated fractionation schedules (ARCON), since for some tumour types, acute and chronic hypoxia as well as treatment protraction may prejudice the outcome of radiotherapy. The tolerable dose of nicotinamide and the optimal interval for administration need to be ascertained. AIM: Full pharmacokinetic profiles of nicotinamide concentrations in plasma were analyzed repeatedly in 15 patients to determine the inter- and intra-patient variability in peak plasma concentrations and the optimum times for administering nicotinamide as a radiosensitizer. METHODS: Nicotinamide (Nicobion) was administered in tablet form to patients with advanced head and neck and non-small cell lung carcinomas. A standard 6 g dose was given regardless of body weight after an overnight fast and at least 30 min before breakfast. In 15 patients, blood samples were taken prior to and 1, 2, 4, 6, 8, 12 and 24 h after administration of the drug. This full profile was determined on two to four occasions for the head and neck cancer patients and on two occasions for the lung cancer patients. For each profile, the maximum concentration of nicotinamide (Cmax), time to peak plasma concentration (Tmax), elimination half-lives (t1/2) and area under the curve (AUC) were determined. Compliance was recorded and nausea and vomiting were graded on a 0-3 scale. Complete profiles of the five major metabolites were also obtained. RESULTS: In the 48 complete sets of blood samples, peak plasma concentrations ranged from 787 to 2312 nmol/ml with a median value of 1166 nmol/ml. The peak plasma concentration was achieved at 1 h in only 54% of the pharmacokinetic profiles, but at this time 92% of the profiles had already exceeded the target concentration of 700 nmol/ml, the level required in the mouse for tumour radiosensitization. The median t1/2 for all 15 cases was 9.3 h, with minimum and maximum values of 4.2 and 26.8 h. The highest concentrations of nicotinamide metabolites were found to be the N-oxide, 2-pyridone and 1-methylnicotinamide. The toxicity (nausea and vomiting) was scored and found not to be correlated with any of the pharmacokinetic parameters. CONCLUSIONS: The plasma concentrations considered necessary to radiosensitize can easily be exceeded with a dose of 6 g taken as 12 x 500 mg in tablet form; 700 nmol/ml was achieved in all patients and apparently would have been achieved in most even with a considerable reduction in dose. An adequate time between administration and radiotherapy appeared to be 1 h with this drug formulation for 92% of the profiles.

Involvement of oxygen free radicals in ischaemia-reperfusion injury to murine tumours: role of nitric oxide.

Ischaemia-reperfusion (I/R) injury is a model system of oxidative stress and a potential anti-cancer therapy. Tumour cytotoxicity follows oxygen radical damage to the vasculature which is modulated by tumour production of the vasoactive agent, nitric oxide (NO.). In vivo hydroxylation of salicylate, to 2,3- and 2,5-dihydroxybenzoate (DHBs), was used to measure the generation of hydroxyl radicals (OH.) following temporary vascular occlusion in two murine tumours (with widely differing capacity to produce NO.) and normal skin. Significantly greater OH. generation followed I/R of murine adenocarcinoma CaNT tumours (low NO. production) compared to round cell sarcoma SaS tumours (high NO. production) and normal skin. These data suggest that tumour production of NO. confers resistance to I/R injury, in part by reducing production of oxygen radicals and oxidative stress to the vasculature. Inhibition of NO synthase (NOS), during vascular reperfusion, significantly increased OH. generation in both tumour types, but not skin. This increase in cytotoxicity suggests oxidative injury may be attenuation by tumour production of NO.. Hydroxyl radical generation following I/R injury correlated with vascular damage and response of tumours in vivo, but not skin, which indicates a potential therapeutic benefit from this approach.

Indolequinone antitumor agents: reductive activation and elimination from (5-methoxy-1-methyl-4,7-dioxoindol-3-yl)methyl derivatives and hypoxia-selective cytotoxicity in vitro.

A series of indolequinones bearing a variety of leaving groups at the (indol-3-yl)methyl position was synthesized by functionalization of the corresponding 3-(hydroxymethyl)indolequinone, and the resulting compounds were evaluated in vitro as bioreductively activated cytotoxins. The elimination of a range of functional groups-carboxylate, phenol, and thiol-was demonstrated upon reductive activation under both chemical and quantitative radiolytic conditions. Only those compounds which eliminated such groups under both sets of conditions exhibited significant hypoxia selectivity, with anoxic:oxic toxicity ratios in the range 10-200. With the exception of the 3-hydroxymethyl derivative, radiolytic generation of semiquinone radicals and HPLC analysis indicated that efficient elimination of the leaving group occurred following one-electron reduction of the parent compound. The active species in leaving group elimination was predominantly the hydroquinone rather than the semiquinone radical. The resulting iminium derivative acted as an alkylating agent and was efficiently trapped by added thiol following chemical reduction and by either water or 2-propanol following radiolytic reduction. A chain reaction in the radical-initiated reduction of these indolequinones (not seen in a simpler benzoquinone) in the presence of a hydrogen donor (2-propanol) was observed. Compounds that were unsubstituted at C-2 were found to be up to 300 times more potent as cytotoxins than their 2-alkyl-substituted analogues in V79-379A cells, but with lower hypoxic cytotoxicity ratios.

Oxidative denitrification of the antitumour drug hydroxyguanidine.

The oxidative denitrification of the antitumour agent hydroxyguanidine (HOG) has been investigated by radiolysis methods and EPR spectroscopy. The azide radical (N3.), a model one-electron oxidant, reacts with HOG with the rate constant 5.1 x 10(9) dm3 mol(-1) s(-1) to yield the guanidino carbon-centred radical (HOG.) which rapidly eliminates nitric oxide (k = 3.1 x 10[3] s[-1]) with the concomitant formation of urea. The HOG. undergoes conjugation with molecular oxygen to form a peroxyl radical (HOGOO.) with a rate constant 8.8 x 10(8) dm3 mol(-1) s(-1). The HOGOO. radical also eliminates nitric oxide but may act as a precursor to the peroxynitrite (ONOO-) ion. The oxidation of HOG by the dibromide radical (Br2.-) was found to release nitric oxide with a yield of 95% relative to Br2.- as determined from the combined yields of inorganic nitrite, nitrate and a HOG/nitric oxide-adduct. This study provides a possible mechanistic basis for the oxidative denitrification of HOG which may contribute to the observed toxicity of the drug both in vitro and in vivo and for the oxidation of nonphysiological hydroxyguanidines to NO. via nitric oxide synthase-independent pathways.

The role of nitric oxide in cancer. Improved methods for measurement of nitrite and nitrate by high-performance ion chromatography.

The short lifetime of nitric oxide (NO) in vivo impedes its quantitation directly; however, the determination of nitrite and nitrate ions as the end-products of NO oxidation has proven a more practical approach. High-performance ion chromatographic analysis of nitrite in biological fluids is hampered by the large amount of chloride ion (up to approximately 100 mmol/l) which results in insufficient peak resolution when utilizing conductimetric detection. Analysis of both anions in small sample volumes is also constrained by the need to minimise sample handling to avoid contamination by environmental nitrate. We report a means to remove Cl- ions from small sample volumes using Ag+ resin which facilitates quantitation of either nitrite and nitrate anions in biological samples, using silica or polymer based ion-exchange resins with conductimetric or electrochemical and spectrophotometric detection. Including a reversed-phase guard column before the anion-exchange guard and analytical column also greatly extends column lifetime.

Administration of nicotinamide during a five- to seven-week course of radiotherapy: pharmacokinetics, tolerance, and compliance.

BACKGROUND AND PURPOSE: Nicotinamide was administered daily as a liquid formulation to head and neck cancer patients receiving a 5- to 7-week course of radiotherapy. The pharmacokinetics, compliance, and tolerance of this drug formulation were studied. MATERIALS AND METHODS: Blood samples were drawn and nicotinamide levels determined in 40 head and neck cancer patients. On the first treatment day serial samples were obtained followed by daily samples at the time of irradiation during the first and last full weeks of the treatment. Side-effects of nicotinamide were monitored. RESULTS: In all patients peak concentrations greater than 700 nmol/ml could be obtained 0.25-3 h (mean 0.83 +/- 0.73 h) after drug intake. During the first week of treatment plasma levels at the time of irradiation were adequate in 82% of the samples. This decreased to 59% in the last week of treatment which can be partly attributed to reduced compliance. The most important side-effect of nicotinamide was nausea with or without vomiting occurring in 65% of the patients. Severe side-effects were associated with high plasma concentrations over subsequent days. Tolerance improved after a 25% reduction of dose in six of seven patients but plasma levels at the time of irradiation fell below 700 nmol/ml in four out of six of these patients. CONCLUSIONS: Peak plasma concentrations above the 700 nmol/ml level were obtained in all patients but these concentrations could not be reproduced during the entire course of the treatment in a significant portion of the subjects. Side-effects of nicotinamide are associated with plasma concentrations and tolerance can be improved by a moderate reduction of dose.

Metabolic and clonogenic consequences of ischaemia reperfusion insult in solid tumours.

Tumour cell survival is intimately related to blood vessel function and so the tumour vasculature represents a novel target for cancer therapy. We have investigated a murine tumour model in which a metal clamp was used to occlude the vascular supply temporarily and then removed to allow reperfusion. This allows the study of ischaemia-reperfusion as a model system for investigating tumour response to metabolic and oxidative stress. Recent studies have shown that prolonged reduction of tumour blood flow results in a deterioration of the hypoxic and acidic microenvironment found within tumours which leads to cytotoxicity. This cytotoxicity is dramatically enhanced if these cells are subsequently reperfused. It was the aim of the present study to determine the relative contribution of cytotoxicity occurring during the ischaemic period and that occurring during reperfusion. Although significant reductions in tumour energy status were induced during the clamping period itself, these were poorly correlated with the degree of tumour cytotoxicity. Relative vascular perfusion, measured using a radiolabelled tracer, remained significantly depressed below the control value following clamp removal. The degree of recovery of perfusion was also dependent upon the clamp duration. Relative tumour perfusion at 1 h after clamp removal was 70.1 +/- 14.6 and 50.5 +/- 6.3% of control values after a 1 or 3 h clamp, respectively, and showed no significant further increase when measured at 24 h after clamp removal. Tumour cytotoxicity following ischaemia reperfusion insult was modulated by administering the anti-oxidant enzymes superoxide dismutase or catalase intravenously just before clamp removal. These enzymes are restricted to the vascular compartment, where it is proposed that they modulate the concentration of oxygen free radicals released during reperfusion and by neutrophil oxidative burst. Reperfusion injury to the tumour was enhanced by administration of an inhibitor of nitric oxide synthase, nitro-L-arginine, probably owing to enhanced neutrophil adhesion and oxidative burst. Conversely, reperfusion injury to the tumour was reduced by administration of a nitric oxide donor, diethylamine nitric oxide. The murine model reported in this paper shows that ischaemia-reperfusion damage mediated by oxygen free radical formation provides a model system for investigating tumour response to oxidative stress at the level of the vascular endothelium.

The relationship between extracellular lactate and tumour pH in a murine tumour model of ischaemia-reperfusion.

We have studied the relationship between extracellular lactate (LACTe) and extracellular pH (pHe) in murine tumours after vascular occlusion (clamping) followed by reperfusion. In tumours occluded at ambient room temperature, LACTe, measured by microdialysis, increased linearly with time and correlated strongly with the acidification of the extracellular compartment (r=0.97, P<0.03, n=4). Significant decrease in LACTe was evident following removal of occlusion at room temperature and is consistent with vascular reperfusion. Occlusion at 35 degrees C, i.e. to maintain tumour temperature during occlusion, resulted in an initial increase in LACTe, which mirrored a rapid reduction in pHe. However further reductions in pHe occurred without increase in LACTe. During vascular occlusion, tumour adenine nucleotide pool decreased and AMP accumulated. AMP subsequently decreased in the 35 degrees C group and this may contribute to the observed differences in accumulation of LACTe, and capacity to recover from vascular occlusion, between the two treatment groups. These data show that extracellular lactate concentration is a good predictor for tumour pH when adequate energy sources are available within the tumour. However, under conditions of more severe stress, resulting in abolition of primary energy stores and cell death, the pHe continues to decline in the absence of a corresponding accumulation of extracellular lactate. This emphasizes the fact that other processes, apart from lactate production, can contribute to reduction in extracellular pH.

Accelerated radiotherapy, carbogen and nicotinamide (ARCON) in locally advanced head and neck cancer: a feasibility study.

BACKGROUND AND PURPOSE: ARCON (Accelerated Radiotherapy, CarbOgen, Nicotinamide) achieves a large therapeutic gain in rodents. A phase I/II study was therefore undertaken to determine its feasibility in patients with locally advanced head and neck cancer. MATERIALS AND METHODS: The accelerated regime CHART was used in 35 patients given carbogen and/or nicotinamide with 11 small volume fractions. Eight patients received carbogen, 12 received nicotinamide and 15 were treated with ARCON. Treatment compliance, side-effects and acute mucositis were monitored in all cases. RESULTS: All patients underwent CHART as intended. In the 23 patients receiving carbogen, two failed to complete treatment. Compliance with nicotinamide was much lower. Out of 25 patients, only 52% received 10-11 doses of the 80 mg/kg/day of the drug. The most common side-effect was nausea and vomiting, which responded to standard anti-emetics in almost half of the patients. Historical comparisons with the CHART head and neck trials indicate that there was no increase in the severity of acute mucositis in any of these patients. Although the observation period is not sufficiently long to be definitive (median 20 months) there is no evidence of an increase in late normal tissue reactions. CONCLUSIONS: ARCON using CHART as the radiotherapy protocol is feasible in patients with advanced head and neck cancer. However, we are concerned about the low compliance rate in our patients, which is far lower than that reported elsewhere. The implications are discussed together with identifying strategies for increasing compliance.

Reduced capacity of tumour blood vessels to produce endothelium-derived relaxing factor: significance for blood flow modification.

The effect of nitric oxide-dependent vasodilators on vascular resistance of tumours and normal tissue was determined with the aim of modifying tumour blood flow for therapeutic benefit. Isolated preparations of the rat P22 tumour and normal rat hindlimb were perfused ex vivo. The effects on tissue vascular resistance of administration of sodium nitroprusside (SNP) and the diazeniumdiolate (or NONO-ate) NOC-7, vasodilators which act via direct release of nitric oxide (NO), were compared with the effects of acetylcholine (ACh), a vasodilator which acts primarily via receptor stimulation of endothelial cells to release NO in the form of endothelium-derived relaxing factor (EDRF). SNP and NOC-7 effectively dilated tumour blood vessels after preconstriction with phenylephrine (PE) or potassium chloride (KCl) as indicated by a decrease in vascular resistance. SNP also effectively dilated normal rat hindlimb vessels after PE/KCl constriction. Vasodilatation in the tumour preparations was accompanied by a significant rise in nitrite levels measured in the tumour effluent. ACh induced a significant vasodilation in the normal hindlimb but an anomalous vasoconstriction in the tumour. This result suggests that tumours, unlike normal tissues are incapable of releasing NO (EDRF) in response to ACh. Capacity for EDRF production may represent a difference between tumour and normal tissue blood vessels, which could be exploited for selective pharmacological manipulation of tumour blood flow.

Nicotinamide pharmacokinetics in humans: effect of gastric acid inhibition, comparison of rectal vs oral administration and the use of saliva for drug monitoring.

The effect of inhibiting gastric acid secretion on nicotinamide pharmacokinetics was studied in five volunteers with the intent of reducing the large variations observed previously in the time to and magnitude of peak plasma concentrations. Plasma levels were determined using a standard high-performance liquid chromatography (HPLC) method after an oral dose of 3 g of nicotinamide either alone or preceded by pretreatment with omeprazole. Suppression of gastric acid production had no significant effect on the rate of uptake or on the peak levels achieved. To bypass gastric acidity, the rectal route was also assessed using a suppository in four volunteers and one patient undergoing radiotherapy. Absorption was slow and variable and much lower plasma levels were observed than after oral dosing. Thus, no improvement in the pharmacokinetics of nicotinamide was observed using either of these two approaches. Parallel estimations were made using a novel and non-invasive method for monitoring nicotinamide pharmacokinetics in saliva. A large and variable fraction of the total amount of nicotinamide-related material in saliva was found to be nicotinic acid, a metabolite not normally found in human plasma. This conversion was inhibited by the use of a chlorhexidine mouthwash, indicating that the oral flora was responsible for its production. The time to peak levels of nicotinamide or of nicotinamide plus nicotinic acid in saliva correlated well with that in plasma. However, peak concentrations for nicotinamide alone were significantly lower than in plasma, and very variable, whereas for nicotinamide plus nicotinic acid saliva levels were 20-30% higher, but more consistent. Although there are some practical difficulties in quantitatively handling saliva, the method is very useful for monitoring nicotinamide pharmacokinetics and for assessment of compliance with nicotinamide treatment.

Chemical properties which control selectivity and efficacy of aromatic N-oxide bioreductive drugs.

Pulse radiolysis was used to generate radicals from one electron reduction of 1,2,4-benzotriazine-1,4-dioxides (derivatives of tirapazamine), and of imidazo [1,2-a]quinoxaline-4-oxides (analogues of RB90740), which have selective toxicity towards hypoxic cells. Radicals from the mono N-oxides (from the latter compounds) react with oxygen approximately 10-40 times faster than does the tirapazamine radical. Radicals from the tirapazamine analogues studied react with oxygen up to approximately 10 times slower than tirapazamine radicals. The quinoxaline N-oxide radicals are involved in prototropic equilibria with pK(a) values (5.5 to 7.4) spanning that reported for tirapazamine (6.0). Generation of radicals radiolytically in the presence of H donors (formate, 2-propanol, deoxyribose) indicate a chain reaction ascribed to H abstraction by the drug radical. The protonated drug radical is much more reactive than the radical anion (H abstraction rate constant approximately equal to 10(2) - 10(3) dm3 mol-1 s-1). Chain termination is ascribed to drug radical-radical reactions, i.e. radical stability in anoxia, with rate constants 2k approximately equal to 1 x 10(7) to 2 x 10(8) dm3 mol-1 s-1 at pH approximately 7.4. Estimates of the reduction potentials of the drug-radical couples in water at pH 7 for two of the mono-N-oxides were in the range-0.7 to 0.8 V vs NHE at pH 7.

Nitric oxide involvement in the toxicity of hydroxyguanidine in leukaemia HL60 cells.

The free-radical intermediates and the stable products formed on one-electron oxidation of hydroxyguanidine (HOG) were investigated in order to suggest a mechanistic basis for HOG-induced cytotoxicity and cytostasis in leukaemia HL60 cells. The azide radical (generated radiolytically) reacted with HOG to produce a carbon-centred radical which in the absence of oxygen decays by a first-order process (k = 3.2 x 10(3) s-1) to yield nitric oxide (NO) and urea. Although the HOG radical reacts rapidly with oxygen (rate constant for O2 addition, k = 4.2 x 10(8) dm3 mol-1 s-1) this neither prevented the elimination of NO. nor generated alternative nitrogen oxides (e.g. peroxynitrite) capable of contributing to cellular oxidative stress. The detection of NO. in HL60 cells corroborated mechanistic studies that oxidative denitrification of HOG does not require catalysis by nitric oxide synthase. Quantitation of NO. by electron paramagnetic resonance (EPR) spectroscopy (utilising a NO. -selective probe) shows higher amounts of NO. under anoxic conditions, reflecting competition for NO. with molecular oxygen in oxic cells. Inhibition of cytochrome P450 and myeloperoxidase activity decreased NO. production thereby identifying these enzyme systems as capable of oxidizing HOG in vitro. A correlation exists between the intracellular levels of NO. with both the cytotoxic and cytostatic effects of HOG within HL60 cells. A higher toxicity was observed with hypoxic than with oxic cells. The lower levels of NO. associated with aerobic conditions caused a G1 --> S block in the cell cycle which under anoxia potentiated NO. -induced apoptotic cell death.

Oxidative denitrification of N omega-hydroxy-L-arginine by the superoxide radical anion.

The superoxide radical anion (O2-.) produced during the catalytic activity of nitric oxide synthase (NOS) and cytochrome P-450 has been implicated in the oxidative denitrification of hydroxyguanidines ( > C = NOH). The reactivity of the radiolytically generated O2-. radical with N omega-hydroxy-L-arginine (NHA) is pH dependent and appears to parallel the prototropic equilibrium of the hydroxyguanidino group ( > C = NOH reversible > C = NO(-)+H+; pK = 8). The N omega-hydroxyguanidino group is more reactive towards O2-. when deprotonated but exhibits negligible reactivity when protonated. Based on a model, the rate constant for the reaction of the O2-. with NHA was estimated as kappa (O2-.+ > C = NO-) approximately 200-500 M-1.s-1, which is probably too low to compete with O2-. reactions with NO- or superoxide dismutase, which occur many orders of magnitude faster. The oxidative elimination of NO from NHA by O2-. was not accompanied by the formation of L-citrulline. Since only 21% of NHA will exist in the deprotonated > C = NO- form at physiological pH, it is unlikely that oxidative denitrification of NHA by cytochrome P-450 or NOS-derived O2-. radicals will prove a major free-radical pathway to NO. and L-citrulline.

Nicotinamide as a repair inhibitor in vivo: studies using single and fractionated X-ray doses in mouse skin and kidneys.

Inhibitors of adenosine diphosphoribosyl transferase, like nicotinamide, 3-aminobenzamide and other analogues, can inhibit repair of radiation-induced sublethal and/or potentially lethal damage in some in vitro systems. Therefore, we have tested the effect of nicotinamide on repair parameters in vivo in two rodent normal tissues. In skin, the sensitivity to dose fractionation (1, 2, 5 or 10 X-ray fractions in 5 days) was monitored by defining the alpha/beta ratio in the presence or absence of nicotinamide (0.5 mg g-1) in air or carbogen. Pre- and postirradiation sensitization were investigated using an X-ray schedule of 5 fractions/5 days in carbogen alone or combined with nicotinamide given 1 h before, immediately after or 8 h after irradiation. Also, changes in the steepness of the underlying X-ray survival curve for the target skin clonogens, reflected by a change in the alpha/beta ratio, were investigated using the neutron top-up design. Underlying survival curves for oxygen +/- nicotinamide were obtained over the X-ray dose range 2.5 to 25 Gy, by administering single X-ray doses and following these with single top-up doses of d(4)-Be neutrons. Finally, in mouse kidney, recovery half-times (t1/2) were obtained by determining the time-dependent disappearance of X-ray damage using a split-dose design of two 6-Gy fractions separated by an interval which varied from 0 to 48 h and followed by two top-up doses from a neutron beam. No increase in alpha/beta for epidermal damage was seen with nicotinamide alone and, although sensitization was observed when the drug was given 1 h before irradiation, no postirradiation sensitization was detected. In kidney, there was no significant difference in the proportion of total repairable damage or in the half-life of recovery between treatments given with or without nicotinamide. Therefore, no decrease in normal tissue tolerance should be observed with the use of nicotinamide in clinical radiotherapy resulting either from reduced sparing with dose fractionation or from an increase in residual damage when shortening the interfraction interval. Finally, unless repair of radiation damage in normal tissues in vivo differs markedly from that of tumors, it is unlikely that the large sensitization seen in rodent tumors at 1.5 to 2 Gy per fraction, with carbogen and nicotinamide, can be attributed to nicotinamide acting as a repair inhibitor.

Scavenging of nitrogen dioxide, thiyl, and sulfonyl free radicals by the nutritional antioxidant beta-carotene.

Mechanisms of free radical scavenging by the nutritional antioxidant beta-carotene have been investigated by pulse radiolysis. Free radicals, which can initiate the chain of lipid peroxidation, including nitrogen dioxide (NO2.), thiyl (RS.), and sulfonyl (RSO2.) radicals, are rapidly scavenged by beta-carotene. Absolute rate constant k[NO2. + beta-carotene] = (1.1 +/- 0.1) x 10(8) m-1 s-1 and for the glutathione thiyl radical k[GS. + beta-carotene] = (2.2 +/- 0.1) x 10(8) m-1 s-1 have been determined. The mechanisms however are mutually exclusive, the former involving electron transfer to generate the radical-cation [ beta-carotene]+. and the latter by radical-addition to generate an adduct-radical [RS... beta-carotene].. Rate constants for thiyl radical-addition reactions vary from 10(6) to 10(9) m-1 s-1 and correlate with the lipophilicity of the thiyl radical under study. Sulfonyl radicals undergo both electron abstraction, [ beta-carotene]+. and radical-addition, [RSO2... beta-carotene]. in an approximate 3:1 ratio. The beta-carotene radical-cation and adduct-radicals are highly resonance stabilized and undergo slow bimolecular decay to non-radical products. These carotenoid-derived radicals react differently with oxygen, a factor which is expected to influence the antioxidant activity of beta-carotene within tissues of varying oxygen tension in vivo.