Free radicals in quinone containing antitumor agents: The nature of the diaziquone (3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone) free radical

The enzymatically generated free radical of the antitumor agent diaziquone is analyzed with the help of two analogs where either the aziridine rings (RQ14) or the carboethoxyamino groups (RQ2) were substituted by chlorine atoms. The hyperfine couplings observed in the diaziquone free radical are due to the nitrogens in the aziridine group. Unresolved coupling and hindered rotation contribute to line broadening. We find that diaziquone free radicals are more stable than RQ14 but less stable than RQ2 free radicals. The reason for this is that the carboethoxyamino groups make the aromatic ring unstable, while the aziridines contribute to its stability. The free radical observed in diaziquone is in all probability that of the parent compound and not that of an intermediate metabolite.     

Electron spin resonance of electrochemically generated free radicals from diaziquone and its derivatives

The one-electron electrochemical reduction of diaziquone (AZQ) and 12 analogs is analyzed using ESR spectroscopy and cyclic voltammetry. The hyperfine coupling constants arising from the interaction of the unpaired electron with the aziridine nitrogen nuclei fall within 1.20 and 2.26 G. Smaller couplings are observed arising from the protons and nitrogens in the carboethoxyamino groups. The in vitro activity of AZQ and its analogs is examined. Methyl groups in the aziridine rings increase the activity of some analogs. In the absence of aziridines, a chloroquinone compound with only carboethoxyamino groups was surprisingly active. This compound has a more positive cathodic peak than diaziquone.     

Mechanism(s) of bioreductive activation. The example of diaziquone (AZQ)

Bioreduction in the activation of diaziquone (2,5-diaziridinyl-3,6-bis (carboethoxyamino)-1,4-benzoquinone) has been investigated by exploring its reduction by whole cells, rat liver microsomes and purified enzymes. The mechanism of bioreduction was further investigated by exploring the chemical and electrochemical reduction of diaziquone as well as its photochemistry. Reduced diaziquone (by several means) was then tested for activity against parent compound. It appears that reduced diaziquone in most cases is more active than the oxidized form. Diaziquone redox cycles, but it is easily reduced to the hydroquinone which oxidizes to the semiquinone yielding free radicals under aerobiosis. The most probable mechanism of action is that of bioreductive alkylation where the alkylating aziridines are protonated after reduction facilitating the opening of the aziridine rings and thus alkylation.     

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.     

Mechanism(s) for the metabolism of mitoxantrone: electron spin resonance and electrochemical studies

Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (-0.79 V) when compared to diaziquone (-0.36 V) and Adriamycin (-0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.     

On the respiratory quotient (RQ) of termites (Insecta: Isoptera)

The respiratory quotient (RQ) at 28 degrees C was determined by Warburg manometry in 23 species of termites from the Mbalmayo Forest Reserve (Cameroon) and three sub-tropical species cultured under laboratory conditions in the U.K. or freshly collected in Australia. The data are tabulated with other recently reported RQs (determined by manometry or GC) and with measured CH(4) emission rates to provide a survey of 29 species covering both lower and higher termites in all major trophic (functional) categories. In all species, except the wood-feeding Coptotermes acinaciformis and the soil-feeding Cubitermes fungifaber, the observed mean values (with manometry corrected for known fluxes of H(2) and CH(4)) were at or well above 1.00. Soil-feeding forms (except C. fungifaber) generally showed a high apparent RQ (not corrected for H(2)), with nine species (out of 13) above 1.20 and six species above 1.30. Well-replicated laboratory experiments with Reticulitermes lucifugus showed that there was a tendency for RQ to fall with time over a 4-h incubation, although remaining greater than 1.00.The observed RQs are consistent with carbohydrate being the principal substrate supporting respiration in all trophic and taxonomic categories, with little or no contribution from the degradation of lignin or other polyaromatic materials. However, in many species (especially soil-feeders), the observed RQ is greater than that expected from known fluxes of O(2), CO(2) and CH(4) on the assumption that carbohydrate is the respiratory substrate. This presupposes that there is a large hydrogen sink (additional to CH(4) production), possibly the emission of H(2) gas, and/or the existence of unresolved digestive mechanisms or electron routings. Uncertainties in the use of manometry with termites are discussed.     

The chemical reduction of diaziquone: products and free radical intermediates

Sodium borohydride reduced diaziquone (AZQ) can cause cross-links between DNA molecules, between DNA and proteins and cause single- and double-strand DNA breaks. In order to understand these effects better, we investigated the reduction of diaziquone by borohydride, and looked at reaction products. We found that a major product was formed during the oxidation of the colorless 2-electron reduced AZQ, and that this product was a monoaziridinyl quinone. We interpret this result to mean that both the leaving aziridine as well as the remaining one can alkylate. This mode of alkylation does not explain cross-links which may occur by a different mechanism requiring simultaneous opening of the aziridine rings. Most of the antitumor activity of borohydride reduced AZQ is probably exerted during the oxidation of the 2-electron reduced AZQ (AZQH2).     

Mechanism(s) involved in meat mutagen formation and inhibition.

The Maillard reaction, which involves Amadori rearrangement as a key step, also results in sugar fragmentation and free radical formation. The imidazoquinoline meat mutagens (2-amino-3-methylimidazo[4,5-f]-quinoline, or IQ, and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, or MeIQ) are formed from a reaction mixture containing alkylpyridine free radicals and creatinine. The imidazoquinoxaline meat mutagens (2-amino-3,4-dimethylimidazo[4,5-f]-quinoxaline, or MeIQx, and 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline, or 4,8-DiMeIQx) may be produced by reacting a mixture containing dialkylpyrazine free radicals and creatinine. Two different pathways for free radical formation are proposed. One involves bimolecular ring formation from the enaminol form of the glycoaldehyde alkylimine and is followed by oxidative formation of the free radical. The other pathway involves formation of N,N1-dialkylpyrazinium ions from glyoxal monoalkylimine followed by reduction to produce the free radicals. The respective intermediates (glycoaldehyde alkylimine and glyoxal monoalkylamine) are formed by reacting glycoaldehyde and glyoxal with amino compounds. The glycoaldehyde system reacts faster and produces more free radicals than the glyoxal system. The reactions help to explain the formation of imidazoquinoxaline meat mutagens and their predominance in fried fish and why these mutagens are present in larger quantities in fried ground beef than the imidazoquinoline-type meat mutagens. These two pathways may not be the only mechanisms involved in formation of meat mutagens, but other free radical reactions may also contribute to meat mutagenicity and are mentioned briefly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free radical formation and DNA strand breakage during metabolism of diaziquone by NAD(P)H quinone-acceptor oxidoreductase (DT-diaphorase) and NADPH cytochrome c reductase.

One-electron reduction of diaziquone (AZQ) by purified rat liver NADPH cytochrome c reductase was associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as indicated by ESR spin-trapping studies. Reactive oxygen formation correlated with AZQ-dependent production of single and double PM2 plasmid DNA strand breaks mediated by this system as detected by gel electrophoresis. Direct two-electron reduction of AZQ by purified rat liver NAD(P)H (quinone acceptor) oxidoreductase (QAO) was also associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as detected by ESR spin trapping. Furthermore, PM2 plasmid DNA strand breaks were detected in the presence of this system. Plasmid DNA strand breakage was inhibited by dicumarol (49 +/- 5%), catalase (57 +/- 2.3%), SOD (42.2 +/- 3.6%) and ethanol (41.1 +/- 3.9%) showing QAO and reactive oxygen formation was involved in the PM2 plasmid DNA strand breaks observed. These results show that both one- and two-electron enzymatic reduction of AZQ give rise to formation of reactive oxygen species and DNA strand breaks. Autoxidation of the AZQ semiquinone and hydroquinone in the presence of molecular oxygen appears to be responsible for these processes. QAO appears to be involved in the metabolic activation of AZQ to free radical species. The cellular levels and distribution of this enzyme may play an important role in the response of tumor and normal cells to this antitumor agent.     

Formation of radical cations of aziridines generated by laser flash photolysis.

The radical cations of 1-butyl-trans-2,3-diphenyl aziridine (1), 1-butyl-2-phenyl aziridine (2), 1,2-diphenyl aziridine (3) and 1-(p-methoxyphenyl)-2-phenyl aziridine (4) were generated upon laser flash photolysis in aqueous and aqueous acetonitrile solutions by direct photoionisation as indicated by the broad absorption band of the solvated electron above 550 nm as well.     

In vitro antioxidant studies of Sphaeranthus indicus (Linn)

The free radical scavenging potential of the plant S. indicus was studied by using different antioxidant models of screening. The ethanolic extract at 1000 microg/ml showed maximum scavenging of the radical cation, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) observed upto 41.99% followed by the scavenging of the stable radical 1,1-diphenyl, 2-picryl hydrazyl (DPPH) (33.27%), superoxide dismutase (25.14%) and nitric oxide radical (22.36%) at the same concentration. However, the extract showed only moderate scavenging activity of iron chelation (14.2%). Total antioxidant capacity of the extract was found to be 160.85 nmol/g ascorbic acid. The results justify the therapeutic applications of the plant in the indigenous system of medicine, augmenting its therapeutic value.     

Free Radicals Scavengers Attenuate Platelet-Activating Factor (PAF)-And Endotoxin-Induced Intestinal Myoelectric Disturbances in Rats

Pretreatment with radical scavengers significantly reduced the intestinal myoelectric disturbances following either E. coli endotoxin or platelet-activating factor (PAF) injection in the rat indicating that free radicals might be involved in the intestinal motor alterations observed in endotoxin shock and that PAF acts partially via free radical production. Moreover, dimethylsulfoxide (DMSO) was found to be more effective in inhibiting the endoxotin-induced intestinal motor alterations, than superoxide dismutase (SOD) and allopurinol. BN 52021, a specific PAF antagonist, was able to reduce the effects of endotoxin on intestinal motility, However, when BN 52021 was combined with free radical scavengers, no additive effect was observed. It is concluded that free radicals involved in endotoxin-induced intestinal motility alterations are at least in part produced in response to PAF.     

An electron spin resonance (ESR) study of free radicals formed from hematoxylin on tissue sections after blueing (alkalization)

A free radical signal of 12 G width and g = 2.0045 can be observed in hematoxylin stained tissue blocks and sections. The amount of paramagnetic centres in stained specimens is significantly larger than in unstained ones. After alkalization simultaneously with the colour change the former free radical is detectable in hemalum precipitate and on stained paper strips. After solution of the stain in dioxane and alkalization, a well resolved hyperfine structure could be seen which could be assigned to three different radicals with the same g value as observed in the rigid matrix (tissue and paper). Quantitative evaluation of free radical concentration is also carried out for tissue sections.     

An electron spin resonance (ESR) study of free radicals formed from hematoxylin on tissue sections after blueing (alkalization)

Summary A free radical signal of 12 G width and g=2.0045 can be observed in hematoxylin stained tissue blocks and sections. The amount of paramagnetic centres in stained specimens is significantly larger than in unstained ones. After alkalization simultaneously with the colour change the former free radical is detectable in hemalum precipitate and on stained paper strips. After solution of the stain in dioxane and alkalization, a well resolved hyperfine structure could be seen which could be assigned to three different radicals with the same g value as observed in the rigid matrix (tissue and paper). Quantitative evaluation of free radical concentration is also carried out for tissue sections.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

The Involvement of Respiration in Free Radical Processes during Loss of Desiccation Tolerance in Germinating Zea mays L. (An Electron Paramagnetic Resonance Study)

When germinating Zea mays L. seeds are rapidly desiccated, free radical-mediated lipid peroxidation and phospholipid de-esterification is accompanied by a desiccation-induced buildup of a stable free radical associated with rapid loss of desiccation tolerance. Comparison of the electron paramagnetic resonance and electron nuclear double resonance properties of this radical with those of the radical in dried, desiccation-intolerant moss showed that the two were identical. At the subcellular level, the radical was associated with the hydrophilic fraction resulting from lipid extraction. Isolated mitochondria subjected to drying were also found to accumulate an identical radical in vitro. When increasing concentrations of cyanide were used, a significant positive correlation was shown between rates of respiration and the accumulation of the radical in desiccation-intolerant tissues. Another positive correlation was found when rates of O2 uptake by radicles at different stages of germination were plotted against free radical content following desiccation. This indicates that free radical production is closely linked to respiration in a process likely to involve the desiccation-induced impairment of the mitochondrial electron transport chain to form thermodynamically favorable conditions to induce accumulation of a stable free radical and peroxidized lipids. Modulation of respiration using a range of inhibitors resulted in broadly similar modulation of the buildup of the stable free radical. One site of radical generation was likely to be the NADH dehydrogenase of complex I and probably as a direct consequence of desiccation-impaired electron flow at or close to the ubiquinone pool.     

达乌尔黄鼠入眠准备期的体温、代谢率及能量特征

贮脂类动物在冬眠前大量积累脂肪来准备冬眠,并在入眠时迅速降低体温和代谢率。为探究入眠准备期达乌尔黄鼠体温、代谢率、呼吸商及能量代谢的变化,将其入眠准备期分为育肥期、体重高峰期、育肥后期和冬眠前的试降期,使用植入式半导体温度记录元件iButton、开放式代谢仪和改进的代谢笼,监测其体温、代谢率及呼吸商和能量摄入的变化。结果显示:(1)达乌尔黄鼠体温在冬眠前13 - 34 d 开始下降,远早于冬眠但晚于体重高峰期;体重高峰期体温有降低的趋势,持续时间为1 - 3 d;育肥后期体温显著下降,体温日波动幅度增加。(2)体重高峰期的静止代谢率高于育肥期,育肥后期有降低的趋势,试降期最低。(3)呼吸商在体重高峰期先升高,之后迅速衰减;入眠准备期的能量摄入在体重达高峰期前达到最大值。结果表明,达乌尔黄鼠在入眠准备期,其体温和代谢率已开始降低,能源物质已开始转变;体重高峰期可能是达乌尔黄鼠入眠的一个转折点或启动入眠的开关。     

Some physicochemical properties of the antitumor drug thiotepa and its metabolite tepa as obtained by density functional theory (DFT) calculations

Density functional theory (DFT) using the B3LYP functional was applied to elucidate the molecular properties of the antitumor drug thiotepa and its main metabolite tepa. Aqueous solvent effects were introduced using the conductor-like polarizable continuum model (CPCM). The protocol for calculating the pK _a values obtained with different cavity models was tested on a series of aziridine and phosphoramide compounds. An efficient computational scheme has been identified that uses the CPCM model of solvation with a universal force field (UFF) cavity. The method has been used to evaluate the basicities of thiotepa and its metabolite. Our calculations show that the basicities of the aziridine moiety of thiotepa and tepa are dramatically reduced compared to free aziridine, indicating that highly acidic media are needed to produce substantial yields of the N-protonated form of the drug. Finally, the mechanisms of reaction of the drug and its metabolite are discussed based on our theoretical results. The calculations reproduce the experimental trends very satisfactorily.     

塔里木兔种群遗传结构的初步探讨

塔里木兔作为中国特有种, 其种群遗传结构直到现在还未知。我们从采自于新疆维吾尔自治区3 个种群的25 号样品中扩增得到529 bp 线粒体DNA 控制区片段, 共发现存在76 个多态性位点。全部样品中定义了19 个单倍型, 每个种群都显示了很高的单倍型多态性。进一步分析表明, 3 个种群存在明显的遗传分化和较低的基因流。通过错配分布和Fu’s 中性检验分析发现, 阿克苏(AKS) , 若羌(RQ) 和疏勒(SL) 种群相对比较稳定。     

Studies related to antitumor antibiotics. Part V. Reactions of mitomycin C with DNA examined by ethidium fluorescence assay.

The cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, three aspects of mitomycin C action on DNA have been studied: (a) cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. Cross-linking of DNA is determined by the return of fluorescence after a heat denaturation step at alkaline pH's. Under these conditions denatured DNA gives no fluorescence. The cross-linking was independently confirmed by S1-endonuclease (EC 3.1.4.-) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was shown not to be due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship exists between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation is due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggests that the cross-linking event, to which the primary cytotoxic action has been linked, occurs sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, was shown to induce single strand cleavage as well as monoaklylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhibition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggests that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C is largely due to the free radical O2. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C does not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C is able to cross-link DNA in the absence of reduction at pH 4. This is consistent with the postulated cross-linking mechansims.