ESR study of electron transfer reactions between gamma-irradiated pyrimidines, adriamycin and oxygen

Solid pyrimidine nucleic acid bases (cytosine, thymine, and uracil) were gamma-irradiated (50 KGy) and dissolved in deaerated solutions of adriamycin in water and dimethylsulfoxide (DMSO). Analogous experiments using unirradiated pyrimidines as controls were also performed. In water only gamma-irradiated cytosine showed a reaction with the adriamycin yielding a single ESR peak (g = 2.0033) consistent with the adriamycin semiquinone radical. Since the unirradiated cytosine gave no reaction, the result suggests an electron transfer from cytosine radicals (generated by gamma-radiolysis) to adriamycin. In DMSO the three gamma-irradiated and unirradiated pyrimidines reacted with adriamycin yielding the adriamycin semiquinone radical observed by ESR. These results suggest that in DMSO an electron is transferred to adriamycin from the pyrimidine radicals and from the parent pyrimidine molecules. However, the process is on the order of 10(5) times more efficient for the pyrimidine radicals. Superoxide radicals (O2-.) were formed following addition of oxygen to the deaerated DMSO solutions containing adriamycin semiquinone radicals. O2-. was spin trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results show a possible reaction sequence in which an electron transferred to adriamycin, by pyrimidine radicals and parent pyrimidine molecules, is subsequently transferred to dissolved oxygen.     

Pulse radiolysis studies of antitumor quinones: Radical lifetimes,reactivity with oxygen,and one-electron reduction potentials

The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 10⁷m^?1s^?1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E₇¹) were determined and were AZQ, ?168 mV, adrenochrome, ?253 mV, mitomycin C, ?271 mV, adriamycin, ?292 mV, daunomycin, ?305 mV, and anthracenedione, ?348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E₇¹.     

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.     

An electron paramagnetic resonance study of the interactions between the adriamycin semiquinone, hydrogen peroxide, iron-chelators, and radical scavengers.

Anaerobic reduction of hydrogen peroxide in a xanthine/xanthine oxidase system by adriamycin semiquinone in the presence of chelators and radical scavengers was investigated by direct electron paramagnetic resonance and spin trapping techniques. Under these conditions, adriamycin semiquinone appears to react with hydrogen peroxide forming the hydroxyl radical in the presence of chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid. In the absence of chelators, a related, but unknown oxidant is formed. In the presence of desferrioxamine, adriamycin semiquinone does not disappear in the presence of hydrogen peroxide at a detectable rate. The presence of adventitious iron is therefore implicated during adriamycin semiquinone-catalyzed reduction of hydrogen peroxide. Formation of alpha-hydroxyethyl radical and carbon dioxide radical anion from ethanol and formate, respectively, was detected by spin trapping. Both the hydroxyl radical and the related oxidant react with these scavengers, forming the corresponding radical. In the presence of scavengers from which reducing radicals are formed, the rate of consumption of hydrogen peroxide in this system is increased. This result can be explained by a radical-driven Fenton reaction.     

Mechanisms of beneficial effects of probucol in adriamycin cardiomyopathy

Probucol, a lipid-lowering drug, has been shown to offer protection against adriamycin-induced cardiomyopathy. In order to define the mechanism of this protection, we examined changes in antioxidants and lipid peroxidation in hearts as well as lipids in hearts and plasma from rats treated with either adriamycin or adriamycin and probucol with appropriate controls. Any potential free radical quenching as well as growth inhibitory effects of probucol were also examined using Chinese hamster ovary (CHO) cells in culture. In animal model, adriamycin caused a significant depression in glutathione peroxidase and increased plasma and cardiac lipids as well as lipid peroxidation. Probucol treatment modulated adriamycin-induced cardiomyopathic changes and increased glutathione peroxidase and superoxide dismutase activities. In the presence of adriamycin under hypoxic conditions, formation of adriamycin semiquinone radical was detected by ESR. The cell growth in these cultures was also inhibited by adriamycin in a dose-dependent manner. Probucol had no effect on adriamycin-induced growth inhibition as well as formation of semiquinone radicals. It is proposed that probucol protection against adriamycin cardiomyopathy is mediated by increased antioxidants and lipid-lowering without any effect on free radical production.     

Iron-mediated formation of an oxidized adriamycin free radical

Electron paramagnetic resonance studies are reported which demonstrate that the reduction of Fe3+ to Fe2+ by adriamycin results in the formation of an oxidized adriamycin free radical with an EPR signal at g = 2.004. A transient iron-adriamycin free radical complex is also observed at g = 2.34. The free radical is quantitated and its aerobic stability is determined. Observation of the oxidized adriamycin free radical signal confirms that adriamycin donates an electron to the bound Fe3+. In the presence of glutathione the drug-mediated reduction of Fe3+ to Fe2+ is bypassed, and the oxidized adriamycin radical signal is not observed. The oxidized adriamycin radicals and reduced oxygen radicals which are formed are two different mediators, whose relative concentrations could modulate the therapeutic and toxic effects of adriamycin.     

Generation of nitro radical anions of some 5-nitrofurans, 2- and 5-nitroimidazoles by norepinephrine, dopamine, and serotonin. A possible mechanism for neurotoxicity caused by nitroheterocyclic drugs

Catecholamine neurotransmitters such as norepinephrine, dopamine, and related catecholamine derivatives reduce nitroheterocyclic drugs such as nitrofurantoin, nifurtimox, nifuroxime, nitrofurazone, misonidazole, and metronidazole in slightly alkaline solutions. Drugs which contain 5-nitrofurans are reduced at lower pH than drugs which contain 2- and 5-nitroimidazoles. 5-Nitroimidazole derivatives such as metronidazole and ronidazole are known to be more difficult to reduce than 2-nitroimidazole derivatives, due to their lower redox potential. Catecholamines, when reducing nitro drugs, undergo concomitant oxidation to form semiquinone radicals. Both semiquinone radicals and nitro anion radicals formed in a reaction of nitro drug and catecholamine derivative were detected by electron spin resonance spectroscopy. Oxygen consumption studies in solutions containing nitro drug and catecholamine derivative showed that nitro anion radicals formed under aerobic conditions reduce oxygen to form the superoxide radical and hydrogen peroxide. Quinones formed in the reaction of catecholamine and nitro drug were detected by optical spectroscopy. Biosynthetic precursors and some metabolic products of catecholamines were also used in these studies, and they all exhibited reactions similar to catecholamines. Bovine chromaffin granules which synthesize and store catecholamines produced the nitrofurantoin anion radical when intact granules were treated with nitrofurantoin. These radicals formed inside the granules were observed by ESR spectroscopy. The formation of nitrofurantoin radical, semiquinone radicals of catecholamines, and oxygen-derived radicals by chromaffin granules is proposed to cause damage to adrenal medulla, and this process may lead to neurotoxicity.     

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex

A mixture of NADPH and ferredoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O2 and superoxide radical is produced. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine:xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

The production of hydroxyl radicals by adriamycin in red blood cells

Spin trapping of the free radicals formed from the interaction between adriamycin and red blood cells resulted in the formation of a hydroxyl spin adduct. The formation of hydroxyl radicals was found to be inhibited by mannitol. Hemoglobin was found not to be obligatory for the formation of hydroxyl radicals which probably result from the reduction of hydrogen peroxide by adriamycin semiquinone.     

Reactions of Adriamycin with Microsomal Iron and Lipids

Iron plays a central role in oxidative injury, reportedly because it catalyzes superoxide- and hydrogen peroxide-dependent reactions yielding a powerful oxidant such as the hydroxyl radical. Iron is also thought to mediate the cardiotoxic and antitumour effects of adriamycin and related compounds. NADPH-supplemented microsomes reduce adriamycin to a semiquinone radical, which in turn re-oxidizes in the presence of oxygen to form superoxide and hence hydrogen peroxide. During this redox cycling membrane-bound nonheme iron undergoes superoxide dismutase- and catalase-insensitive reductive release. Membrane iron mobilization triggers lipid peroxidation, which is markedly enhanced by simultaneous addition of superoxide dismutase and catalase. The results indicate that : i) lipid peroxidation is mediated by the release of iron, yet the two reactions are governed by different mechanisms; and ii) oxygen radicals are not involved in or may actually inhibit adriamycin-induced lipid peroxidation. Microsomal iron delocalization and lipid peroxidation might represent oxyradical-independent mechanisms of adriamycin toxicity.     

DNA damage by superoxide-generating systems in relation to the mechanism of action of the anti-tumour antibiotic adriamycin

1. A mixture of NADPH and ferrodoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O₂ and superoxide radical is produced. 2. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine: xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. 3. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. 4. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones

Direct and spin-trapping electron spin resonance methods have been used to study the reactivity of semiquinone radicals from the anthracycline antibiotics daunorubicin and adriamycin towards peroxides (hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide). Semiquinone radicals were generated by one-electron reduction of anthracyclines, using xanthine/xanthine oxidase. It is shown that the semiquinones are effective reducing agents for all the peroxides. From spin-trapping experiments it is inferred that the radical product is either OH (from H2O2) or an alkoxyl radical (from the hydroperoxides) which undergoes beta-scission to give the methyl radical. The rate constant for reaction of semiquinone with H2O2 is estimated to be approx. 10(4)-10(5) M-1 X s-1. The reduction does not appear to require catalysis by metal ions.     

Free radical formation by antitumor quinones

Quinones are among the most frequently used drugs to treat human cancer. All of the antitumor quinones can undergo reversible enzymatic reduction and oxidation, and form semiquinone and oxygen radicals. For several antitumor quinones enzymatic reduction also leads to formation of alkylating species but whether this involves reduction to the semiquinone or the hydroquinone is not always clear. The antitumor activity of quinones is frequently linked to DNA damage caused by alkylating species or oxygen radicals. Some other effects of the antitumor quinones, such as cardiotoxicity and skin toxicity, may also be related to oxygen radical formation. The evidence for a relationship between radical formation and the biological activity of the antitumor quinones is evaluated.     

Detection of free radicals during the cellular metabolism of adriamycin

Experiments were conducted to determine which free radicals are generated during the metabolism of adriamycin (ADM) by canine tracheal epithelial (CTE) cells, guinea pig enterocytes, and rat hepatocytes. The technique employed in this study was spin trapping; the spin trap utilized was 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). The spin adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed during the metabolism of ADM by CTE cells. However, the addition of dimethyl sulfoxide to the in vitro system suggested that superoxide is initially spin trapped by the nitrone, and that the adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH) is rapidly bioreduced to afford DMPO-OH. The addition of superoxide dismutase to the system indicated that superoxide generation was primarily intracellular. The adriamycin semiquinone free radical (ADM-SQ) was produced during the metabolism by enterocytes and hepatocytes. The rate of the production of ADM-SQ was enhanced under anaerobic conditions, suggesting that molecular oxygen was responsible for the degradation of this carbon-centered free radical. However, spin trapping of oxygen radicals was not observed; this observation suggests that these reactive intermediates are not produced at concentrations sufficient for detection by spin-trapping experiments.     

The in vitro metabolites of 2,4,6-trichlorophenol and their DNA strand breaking properties

The carcinogenic compound 2,4,6-trichlorophenol (2,4,6-TCP) was incubated with rat liver S-9 fraction. Three metabolites were identified: 2,6-dichloro-1,4-hydroquinone (DHQ), and two isomers of hydroxypentachlorodiphenyl ether (OH-Cl5-DPE). The latter are probably products of microsomal .OH radical attack on the trichlorophenol molecule forming phenoxy free radicals. These would undergo dimerizations with other molecules present in solution. The 2,6-dichloro-1,4-semiquinone free radical was identified by ESR spectroscopy. It is formed at physiological conditions in phosphate buffer at pH 7.2 and 7.8, with a more intensive signal at the more alkaline pH. The formation is probably due to the autoxidation of the corresponding hydroquinone. Incubation of a mixture of metabolites with PM2 DNA at pH 7.2 resulted in single strand breaks. Addition of catalase and dimethylsulfoxide (DMSO) inhibited the DNA strand scission. It was concluded that reactive oxygen species (ROS), produced during the formation of the semiquinone radical, were responsible for the observed DNA damage. The significance of the ROS and the semiquinone free radical is discussed in view of the reported tumorgenicity of 2,4,6-TCP in rats and mice.     

New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.     

Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen

The anticancer drug adriamycin binds iron and these complexes cycle to reduce molecular oxygen (Zweier, J. L. (1984) J. Biol. Chem. 259, 6056-6058). Optical absorption, EPR, and M?ssbauer spectroscopic data are correlated with polarographic O2 consumption and chemical Fe2+ extraction measurements in order to characterize each step in this cycle. Fe3+ binds to adriamycin at physiologic pH forming a complex with an optical absorbance maximum at 600 nm. EPR signals at g = 4.2 and g = 2.01, and a doublet M?ssbauer spectrum with isomer shift delta = 0.57 mm/s and quadrupole splitting delta EQ = 0.74 mm/s are observed indicating that the Fe3+ bound to adriamycin is high spin S = 5/2. Under anaerobic conditions the absorbance maximum at 600 nm decreases with an exponential decay constant = 0.77 h-1, and the EPR and M?ssbauer spectra of Fe3+-adriamycin similarly decrease as the Fe3+ is reduced to EPR silent Fe2+. The Fe2+-adriamycin complex which is formed exhibits a M?ssbauer spectrum with delta = 1.18 mm/s and delta EQ = 1.82 mm/s indicative of high spin Fe2+. As the EPR spectra of Fe3+-adriamycin decrease on reduction of the Fe3+ to Fe2+ a signal of the oxidized adriamycin free radical appears at g = 2.004 with line width of 8 G. On exposure to O2 the absorption maximum at 600 nm, the Fe3+ EPR, and the Fe3+ M?ssbauer spectra all return. Polarographic measurements demonstrate that O2 is consumed and that H2O2 is formed. Addition of high affinity Fe2+ chelators block O2 consumption indicating that Fe2+ formation is essential for O2 reduction. This cycle of iron-mediated O2 reduction can explain the formation of the reactive reduced oxygen and adriamycin radicals which are thought to mediate the biological activity of adriamycin.     

Resistance of paraquat and adriamycin in human breast tumor cells: role of free radical formation

Generation and enhanced detoxification of toxic free radicals by glutathione peroxidase and glutathione transferase in human breast tumor cells have been suggested to play an important role in toxicity and in resistance to adriamycin. We have examined the biochemical basis of paraquat-induced free radical formation and the mechanism of resistance to this agent in human breast tumor cell lines. We have also compared the similarities and differences between adriamycin and paraquat in their mode of free radical formation and tumor cell kill. Anaerobic incubation of paraquat resulted in the formation of the paraquat cation radical in both the sensitive and resistant cells which increased with time and was enhanced by NADPH addition. Our studies show that while both adriamycin and paraquat form hydroxyl radicals (.OH) in these cell lines, adriamycin was 2-3 fold better at reducing oxygen. The formation of .OH was inhibited by exogenously added superoxide dismutase and catalase, indicating the involvement of both superoxide anion radical and hydrogen peroxide. In the adriamycin-resistant cell line, less .OH was formed by each of these drugs. While the .OH appeared to be formed outside by both adriamycin and paraquat in the drug-sensitive cells, experiments using chromium oxalate as a spin-broadening agent suggest that the drug-induced .OH formation in the resistant cells is an intracellular event. The adriamycin-resistant cell line was also cross-resistant to paraquat, suggesting a common mechanism of toxicity for both drugs. However, adriamycin was significantly more toxic (4000-times) to the sensitive cells suggesting that either other mechanisms or site-specific free radical formation are also important in biochemical mechanisms of adriamycin toxicity.     

Free radicals and anticancer drug resistance: Oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors

Certain anticancer agents form free radical intermediates during enzymatic activation. Recent studies have indicated that free radicals generated from adriamycin and mitomycin C may play a critical role in their toxicity to human tumor cells. Furthermore, it is becoming increasingly apparent that reduced drug activation and or enhanced detoxification of reactive oxygen species may be related to the resistance to these anticancer agents by certain tumor cell lines. The purposes of this review are to summarize the evidence pointing toward the significance of free radicals formation in drug toxicity and to evaluate the role of decreased free radical formation and enhanced free radical scavenging and detoxification in the development of anticancer drug resistance by a spectrum of tumor cell types. Studies failing to support the participation of oxyradicals in the cytotoxicity and resistance of adriamycin are also discussed.     

The structural basis for anthracycline antibiotic stimulation of oxygen consumption by HL-60 cells and mitochondria

The effects of anthracyclines on the stimulation of oxygen consumption in the presence of HL-60 cell sonicates, beef heart mitochondria and NADPH cytochrome c reductase were determined as a measure of oxygen radical production. Drug-induced oxygen radical formation in each of these systems was modulated by structural changes in the aglycone as well as in the amino sugar portion of the anthracycline molecule. Cytotoxic potency was not correlated with anthracycline-induced oxygen consumption, suggesting that net oxygen radical production was not the primary factor in tumor cell killing by anthracyclines. In contrast, available data on anthracycline cardiotoxicity appeared to correlate with the drug-induced stimulation of oxygen consumption by beef heart mitochondria, providing support for the premise that drug-induced oxygen radicals formed in the presence of mitochondrial flavoproteins are involved in the adverse effects of anthracyclines on the heart. Cyanomorpholinoadriamycin, an analogue which is 100 to 1000 times more potent than adriamycin (doxorubicin) as an antineoplastic agent, has been shown here and elsewhere to be equivalent to adriamycin in stimulating oxygen radical production by beef heart mitochondria and to produce similar cardiotoxicity at equimolar concentrations. Thus, it appears possible to separate the favorable antitumor activity of adriamycin from its unwanted cardiotoxicity by structural changes such as substitution of the antibiotic by a cyanomorpholino moiety.