A Comparison of the Free Radical Properties of Several Anthracycline Anti-Tumour Drugs and Some of Their Analogues

NADPH consumption and esr spectroscopy have been used to study the rate of formation and signal intensity of free radicals produced by various anthracycline anti-tumour drugs in rat liver microsomal extract. The drugs investigated were Adriamycin, 4'Deoxyadriamycin, Daunorubicin, 4 Demethoxydaun-orubicin and Carminomycin. Pulse radiolysis was also used to determine the ease of reduction of each of the analogues to its semiquinone radical and some kinetic properties of the radicals produced. It is believed that the Occurrence of reactions other than dismutation could be responsible for the shortened lifetimes of the semiquinone radicals observed in biological systems.     

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.     

The reduction of anti-tumour diaziridinyl benzoquinones

The properties of the semiquinone radicals produced for 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), have been investigated. AZQ semiquinone radicals can be produced from the reduction of AZQ by superoxide radicals, whereas BZQ semiquinone radicals are unstable in the presence of oxygen. The one-electron reduction potentials of the couples Q/Q-. at pH 7.0 were determined as -70 +/- 10 mV for AZQ and -376 +/- 15 mV for BZQ. The difference in these potentials is explained. As a consequence of ESR studies on the enzymatically produced radicals, we have considered the factors which determine the detection of ESR signals for reduced quinones produced in a biological system.     

Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light

When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl-1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy.     

Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects.     

Characterization of the Na-dependent respiratory chain NADH:quinone oxidoreductase of the marine bacterium, Vibrio alginolyticus, in relation to the primary Na pump

The Na⁺-dependent respiratory chain NADH: quinone oxidoreductase of the marine bacterium, Vibrio alginolyticus, was extracted from membrane by a detergent, Liponox DCH, and was purified by chromatography on QAE-Sephadex and Bio-Gel HTP. The activity of NADH oxidation was separated into two fractions. The one fraction could react with several artificial electron acceptors including Q-1, but could not reduce ubiquinone and menaquinone such as Q-5 and menaquinone-4, which was called NADH dehydrogenase. The other fraction could reduce Q-5 and menaquinone-4 in addition to the NADH dehydrogenase activity, which was called quinone reductase. The purified NADH dehydrogenase consumed NADH in excess of the amount of Q-1 and the reduced Q-1 (quinol) was not produced at all due to an oxidation-reduction cycle of semiquinone radicals. The quinone reductase, however, consumed NADH with the quantitative formation of quinol on account of a dismutation reaction of semiquinone radicals. Identical to the membrane-bound NADH: quinone oxidoreductase, the quinone reductase specifically required Na⁺ for the activity and was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. The electron transfer in the quinone reductase was formulated in a form of quinone cycle and the dismutation reaction of semiquinone radicals was assigned to be coupled to the Na⁺ pump in the respiratory chain of this organism.     

Radicals involved in photoallergen/protein interactions

Aqueous solutions (pH = 8) of both 3,3'-dimethyl and 4,4'-dimethyl substituted analogues of the photoallergen fentichlor (bis(2-hydroxy-5-chlorophenyl)sulphide) produced stable semiquinone radicals when irradiated with u.v. light (greater than 310 nm). These radicals have been characterised using electron spin resonance techniques: the results confirm the assignment of hyperfine coupling constants for the parent fentichlor radical. The binding of fentichlor to HSA was found to be partly oxygen dependent demonstrating a role for semiquinone type radicals in the binding mechanism. The stoichiometry and specificity of the binding of the dimethyl analogues to soluble proteins were found to be similar to that of fentichlor itself.     

Free radicals and their biological significance: present and future]

Free radicals are usually active species which have unpair electron (S) in molecules or Atomic groups. Of the free radicals, O2- and .OH could easily be produced in mammalian cells, by oxidation and reduction cycle catalyzed by fravoproteins and by iron + H2O2 reaction, respectively. Other free radicals would also be produced in mammalian cell, such as amino acid radicals, semiquinone radicals and flavine radicals etc. In general, free radicals cause cell injury though membrane lipid peroxidation and DNA strand cleavage and some other mechanisms.     

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.     

Reduction of Antitumour Mitosenes in Non-Aqueous and Aqueous Environment. An Electron Spin Resonance and Cyclic Voltammetry Study

Chemical reduction of mitosenes under aerobic conditions in DMSO showed characteristic ESR signals of the mitosene derived semiquinone free radicals. However, these signals diminished strongly upon addition of water to the reaction mixture, indicating a short lifetime of the mitosene semiquinone free radicals under aqueous conditions. In addition, enzymatic one-electron reduction of these mitosenes with either xanthine oxidase or purified NADPH cytochrome P450 reductase under anaerobic conditions showed no signals of the mitosene semiquinone free radicals. Subsequent cyclic voltammetry measurements demonstrated facilitation of the further one-electron reduction of the mitosene semiquinone free radicals in the presence of water in comparison with non-aqueous conditions. The present results strongly suggest that in the presence of water relatively stable hydroquinones are formed upon reduction of mitosenes. Consequently, the steady state concentrations of mitosene semiquinone free radicals will be lowered substantially in aqueous environment. Thus under physiological conditions, two-electron reduction and formation of the mitosene hydroquinone might be important in processes leading to DNA alkylation by these mitosenes.     

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.     

Inhibition of anthracycline semiquinone formation by ICRF-187 (Dexrazoxane) in cells

The formation of semiquinone free radicals of doxorubicin, epirubicin, daunorubicin, and idarubicin was measured by electron paramagnetic resonance (EPR) spectroscopy in hypoxic suspensions of chinese hamster ovary (CHO) cells. The amount of semiquinone produced was in the order idarubicin > doxorubicin > daunorubicin > epirubicin. The idarubicin semiquinone signal was both the fastest to be formed and to decay. Idarubicin, which was the most lipophilic of the anthracyclines studied, also displayed the fastest fluorescence-measured cellular uptake of drug. Thus, it was concluded that semiquinone formation was dependent upon the rate of cellular uptake. Lysed cell suspensions were also shown to be capable of producing the doxorubicin semiquinone in the presence of added NADPH. The cardioprotective agent dexrazoxane (ICRF-187) was observed to decrease the amount of doxorubicin semiquinone observed in cell suspensions. Dexrazoxane also decreased the amount of doxorubicin semiquinone observed in the NADPH-lysed cell suspension mixture. Neither bipyridine nor deferoxamine decreased NADPH-dependent doxorubicin semiquinone formation. These results suggest that dexrazoxane does not decrease doxorubicin semiquinone formation through an iron complex formed from hydrolyzed dexrazoxane. Dexrazoxane may be inhibiting an NADPH-dependent enzyme.     

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.     

Thiol oxidation induced by oxidative action of adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Effect of the microsomal system on interconversions between hydroquinone, benzoquinone, oxygen activation, and lipid peroxidation

Our previous results indicated that cytochrome P450 destruction by benzene metabolites was caused mainly by benzoquinone (Soucek et al., Biochem. Pharmacol. 47 (1994) 2233-2242). The aim of this study was to investigate the interconversions between hydroquinone, semiquinone, and benzoquinone with regard to both spontaneous and enzymatic processes in order to test the above hypothesis. We have also studied the participation of hydroquinone and benzoquinone in OH radicals formation and lipid peroxidation as well as the role of ascorbate and transition metals. In buffered aqueous solution, hydroquinone was slowly oxidized to benzoquinone via a semiquinone radical. This conversion was slowed down by the addition of NADPH and completely stopped by microsomes in the presence of NADPH. Benzoquinone was reduced to semiquinone radical at a significantly higher rate and this conversion was stimulated by NADPH and more effectively by microsomes plus NADPH while semiquinone radical was quenched there. In microsomes with NADPH. both hydroquinone and benzoquinone stimulated the formation of OH radicals but inhibited peroxidation of lipids. Ascorbate at 0.5-5 mM concentration also produced significant generation of OH radicals in microsomes. Neither hydroquinone nor benzoquinone did change this ascorbate effect. On the contrary, 0.1-1.0 mM ascorbate stimulated peroxidation of lipids in microsomes whereas presence of hydroquinone or benzoquinone completely inhibited this deleterious effect of ascorbate. Iron-Fe2+ apparently played an important role in lipid peroxidation as shown by EDTA inhibition, but it did not influence OH radical production. In contrast, Fe3+ did not influence lipid peroxidation, but stimulated OH radical production. Thus, our results indicate that iron influenced the above processes depending on its oxidation state, but it did not influence hydroquinone/benzoquinone redox processes including the formation of semiquinone. It can be concluded that interconversions between hydroquinone and benzoquinone are influenced by NADPH and more effectively by the complete microsomal system. Ascorbate, well-known antioxidant produces OH radicals and peroxidation of lipids. On the other hand, both hydroquinone and benzoquinone appear to be very efficient inhibitors of lipid peroxidation.     

Quinoid redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter.

The health effects of airborne fine particles are the subject of government regulation and scientific debate. The aerodynamics of airborne particulate matter, the deposition patterns in the human lung, and the available experimental and epidemiological data on health effects lead us to focus on airborne particulate matter with an aerodynamic mean diameter less than 2.5 microm (PM(2.5)) as the fraction of the particles with the largest impact in health. In this article we present a novel hypothesis to explain the continuous production of reactive oxygen species produced by PM(2.5) when it is deposited in the lung. We find PM(2.5) contains abundant persistent free radicals, typically 10(16) to 10(17) unpaired spins/gram, and that these radicals are stable for several months. These radicals are consistent with the stability and electron paramagnetic resonance spectral characteristics of semiquinone radicals. Catalytic redox cycling by semiquinone radicals is well documented in the literature and we had studied in detail its role on the health effects of cigarette smoke particulate matter. We believe that we have for the first time shown that the same, or similar radicals, are not confined to cigarette smoke particulate matter but are also present in PM(2.5). We hypothesize that these semiquinone radicals undergo redox cycling, thereby reducing oxygen and generating reactive oxygen species while consuming tissue-reducing equivalents, such as NAD(P)H and ascorbate. These reactive oxygen species generated by particles cause oxidative stress at sites of deposition and produce deleterious effects observed in the lung.     

Feeding on poplar leaves by caterpillars potentiates foliar peroxidase action in their guts and increases plant resistance

Peroxidases (PODs) are believed to act as induced and constitutive defenses in plants against leaf-feeding insects. However, little work has examined the mode of action of PODs against insects. Putative mechanisms include the production of potentially antinutritive and/or toxic semiquinone free radicals and quinones (from the oxidation of phenolics), as well as increased leaf toughness. In this study, transgenic hybrid poplar saplings (Populus tremula × Populus alba) overexpressing horseradish peroxidase (HRP) were produced to examine the impact of elevated HRP levels on the performance and gut biochemistry of Lymantria dispar caterpillars. HRP-overexpressing poplars were more resistant to L. dispar than wild-type (WT) poplars when the level of a phenolic substrate of HRP (chlorogenic acid) was increased, but only when leaves had prior feeding damage. Damaged (induced) leaves produced increased amounts of hydrogen peroxide, which was used by HRP to increase the production of semiquinone radicals in the midguts of larvae. The decreased growth rates of larvae that fed on induced HRP-overexpressing poplars resulted from post-ingestive mechanisms, consistent with the action of HRP in their midguts. The toughness of HRP-overexpressing leaves was not significantly greater than that of WT leaves, whether or not they were induced. When leaves were coated with ellagitannins, induced HRP leaves also produced elevated levels of semiquinone radicals in the midgut. Decreased larval performance on induced HRP leaves in this case was due to post-ingestive mechanisms as well as decreased consumption. The results of this study provide the first demonstration that a POD is able to oxidize phenolics within an insect herbivore’s gut, and further clarifies the chemical conditions that must be present for PODs to function as antiherbivore defenses.     

Electron spin resonance studies of the interaction of oxidoreductases with 2,6-dimethoxy-p-quinone and semiquinone

Previous electron spin resonance studies have demonstrated that the decay of ascorbyl plus semiquinone radicals, produced in an aqueous mixture of ascorbate and 2,6-dimethoxy-p-quinone, is accelerated by ascites cells. This effect was concluded to involve a sulfhydryl-containing NAD(P)H-enzyme, and work on cultured cell lines showed that on neoplastic transformation the activity against the radicals was increased. We show here that at least three disulfide-oxidoreductases are able to quench the radicals in a similar way to that of viable cells. Glutathione reductase (EC 1.6.4.2) in the presence of NADPH and oxidised glutathione, and dihydrolipoamide dehydrogenase (EC 1.8.1.4) with NADH and lipoamide, are found to accelerate the radical decay by reducing the quinone or semiquinone. DT-diaphorase (EC 1.6.99.2) in the presence of NAD(P)H can also achieve this by reducing the quinone directly. Lipoamide dehydrogenase and glutathione reductase are also capable of reducing nitroxide spin labels, a finding considered of relevance to the reported reduction of such spin labels by neuroblastoma cells.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.