Involvement of NADPH: cytochrome P450 reductase in the activation of indoloquinone EO9 to free radical and DNA damaging species.

Evidence suggests that DT-diaphorase is involved in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9 under aerobic conditions. Data also implicate a role for other enzymes including NADPH: cytochrome P450 reductase, especially in low DT-diaphorase tumour cells and under hypoxic conditions. Here, we used purified rat NADPH: cytochrome P450 reductase to provide additional evidence in support of a role for this enzyme in activation of EO9 to generate free radical and DNA-damaging species. Electron spin resonance spectrometry studies showed that NADPH: cytochrome P450 reductase reduced EO9 to a free radical species, including a drug radical (most likely the semiquinone) and reactive oxygen species. Plasmid DNA experiments showed that reduction of EO9 catalysed by NADPH: cytochrome P450 reductase results in single-strand breaks in DNA. The information obtained may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9 and may be useful in the design of future bioreductive drugs.

The safety and efficacy of cisatracurium 0.15 mg.kg(-1) during nitrous oxide-opioid anaesthesia in infants and children.

We studied the neuromuscular and cardiovascular effects of a single, rapidly administered intravenous dose of cisatracurium 0.15 mg.kg(-1) in 27 infants (aged 1-23 months) and 24 children (aged 2-12.5 years). After midazolam premedication, anaesthesia was induced and maintained with thiopental and alfentanil in addition to nitrous oxide in oxygen. Neuromuscular function was monitored by evoked adductor pollicis electromyography. At least 15 min after intubation, each patient received cisatracurium 0.15 mg.kg(-1) over 5 s. Complete neuromuscular blockade was produced by this dose in all but one infant. The mean (SD) onset time of maximal blockade was more rapid in infants [2.0 (0.8) min] than in children [3.0 (1.2) min], p = 0. 0011. The clinical duration of action of cisatracurium (recovery of evoked response to 25% of control) was significantly longer in infants [43.3 (6.2) min] than in children [36.0 (5.4) min], p < 0.0001. Once neuromuscular function started to recover, the rate of recovery was similar in both age groups. Changes in blood pressure and heart rate after the administration of cisatracurium were negligible in both age groups. Cisatracurium, at a dose of 0.15 mg. kg(-1), was effective and well tolerated in infants and children.

Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes.

The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5' end). A 5' RACE reaction was used to sequence the 5' terminus from WEE strain CBA87. The deduced WEE genome was 11508 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28% A, 25% C, 25% G and 22% U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation; a total of 149 nucleotide differences in 8624 bases (1.7% divergence), of which only 28% (42 nucleotides) altered the encoded amino acids. Comparison of deduced nsP1 and nsP4 amino acid sequences from WEE with the corresponding proteins from eastern equine encephalitis virus (EEE) yielded identities of 84.9 and 83.8%, respectively. Previously uncharacterized stem-loop structures were identified in the nontranslated terminal regions. A cDNA clone of the 26S region encoding the structural polyprotein of WEE strain 71V-1658 was placed under the control of a cytomegalovirus promoter and transfected into tissue culture cells. The viral envelope proteins were functionally expressed in tissue culture, as determined by histochemical staining with monoclonal antibodies that recognize WEE antigens, thus, forming the initial step in the investigation of subunit vaccines to WEE.

The symbiotic water mite Unionicola formosa (Acari: Unionicolidae) ingests mucus and tissue of its molluscan host.

Unionicola formosa is a symbiotic water mite that passes most of its life cycle in the mantle cavity of freshwater mussels. Although mites of this genus are often referred to as parasitic, little is known about their nutritional biology. A few species reportedly pierce the gill of a host mussel and ingest tissue or hemolymph. The present study was undertaken to identify possible sources of nutrition for U. formosa. To determine if mites ingested particulate matter in the mucous strand produced by a mussel during feeding, mussels with resident mites were exposed to a suspension of fluorescent microspheres. There was no evidence that U. formosa ingested the beads. Histochemical staining did, however, indicate a mucous material present in the midgut of the mites. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic assays revealed a high molecular weight component, consistent with a mucopolysaccharide, present both in the mussel gill and the mites. Results from western blots and an immunoaffinity binding assay with antibodies against mussel gill tissue and hemolymph also indicated that mites ingested host tissue. Whereas U. formosa probably does not ingest particulate material acquired by its host's suspension feeding, it is apparent that this mite utilizes host mucus, gill tissue, or hemolymph for at least part of its nutrition.

Reduction of the indoloquinone anticancer drug EO9 by purified DT-diaphorase: a detailed kinetic study and analysis of metabolites.

DT-diaphorase has been implicated in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9. Here, we have used a highly purified DT-diaphorase isolated from rat Walker tumour cells to provide unambiguous evidence for the ability of this enzyme to catalyze reduction of EO9 and to provide a more detailed characterization of the reaction. Under the conditions used hypoxia had no effect on the initial rate of this reduction but did effect the nature and stability of metabolites formed. Electron spin resonance (ESR) spectrometry studies showed that DT-diaphorase reduced EO9 to a highly oxygen-sensitive metabolite that is probably the hydroquinone. In the presence of air, this metabolite is auto-oxidized to generate both drug- and oxygen-based radicals. Comproportionation:disproportionation reactions may also be involved in the generation of these radical species. The identification of these metabolites may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9.

Telemedicine and the payment system.

Free medical phone service has become a professional courtesy, but recent changes in the practice environment should prompt a reexamination of this free service as perhaps an anachronism which is getting in the way of progress.

Resistance to oxidants associated with elevated catalase activity in HL-60 leukemia cells that overexpress multidrug-resistance protein does not contribute to the resistance to daunorubicin manifested by these cells.

PURPOSE: It has been recognized that enhanced antioxidant defenses can contribute to the resistance of cancer cells displaying multidrug resistance (MDR) that arises in conjunction with the overexpression of P-glycoprotein (Pgp). The purpose of this study was to determine if the defenses against oxidant stress in MDR human leukemia cells (HL-60/AR) that overexpress multidrug-resistance-associated protein (MRP), but not Pgp, contribute to the mechanism of drug resistance in this cell line. METHODS: HL-60/AR cells were evaluated in comparison with wild-type cells with respect to sensitivity to the oxidants hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (t-BuOOH), the activities and amounts of the antioxidant enzymes catalase and glutathione peroxidase (GSH-Px), and the effects that manipulation of the activities of these enzymes may have on cellular sensitivity to the oxidants and to daunorubicin. We also evaluated the ability of the cells to generate daunorubicin semiquinone free radical as measured by electron spin resonance (ESR) spectroscopy. RESULTS: HL-60/AR cells were > 10-fold resistant to the cytotoxic effects of the H2O2 or t-BuOOH as compared with parental, drug-sensitive HL-60 cells. This phenomenon could be attributed largely to elevated activity and protein levels of catalase in HL-60/AR cells. Furthermore, inhibition of catalase by 3-amino-1,2,4-triazole (AT) diminished the resistance of HL-60/AR to these oxidants by > 80% or > 50%, respectively. Despite these findings, AT was incapable of causing sensitization of HL-60/AR cells to the cytotoxic effects of daunorubicin. We found that the activity and amount of selenium-dependent glutathione peroxidase (GSH-Px) was no greater in HL-60/AR cells than in HL-60 cells. Cultivation of cells in selenium-deficient medium caused a marked reduction in GSH-Px activity in HL-60/AR cells and a profound inhibition of GSH-redox cycling manifested by a decrease in baseline hexose monophosphate shunt activity (HMPS) and markedly blunted stimulation of the HMPS by the oxidant t-BuOOH in both wild-type and resistant cells. These variations in GSH-Px activity and GSH-redox cycling, however, were not associated with an alteration in cellular sensitivity to daunorubicin. The failure of catalase inhibition or selenium manipulation of GSH-Px activity to affect daunorubicin cytotoxicity was not due to the inability of these cells to produce free-radical species of daunorubicin, since ESR studies revealed that the generation of daunorubicin semiquinone free radical by HL-60/AR cells was equal to and, in fact, 3-fold that obtained with HL-60 cells. CONCLUSIONS: In comparison with parental HL-60 cells, MRP-overexpressing HL-60/AR cells have demonstrable alterations in antioxidant defenses that are manifested by cellular resistance to the cytotoxic effects of H2O2 and t-BuOOH and by elevated protein levels and activity of catalase. Whether these alterations are epiphenomena or are related to overexpression of MRP remains to be determined. However, it does appear that the enhanced antioxidant defenses observed in HL-60/AR cells do not contribute to the resistance to daunorubicin manifested by this cell line. Although HL-60/AR cells generate daunorubicin semiquinone free radical to an extent equal to or greater than that observed in HL-60 cells, the failure of alterations in GSH-Px activity or inhibition of catalase to change the sensitivity of HL-60/AR cells to daunorubicin suggests that the cytotoxicity of daunorubicin in these cells in not mediated through H2O2 or other peroxide species detoxified by these enzymes.

Aliphatic amine N-oxides of DNA binding agents as bioreductive drugs.

The DNA binding and cytotoxicity of four intercalating agents, namely bis-alkylamino (-N(CH2)2N(CH3)2) substituted anthraquinone, anthrapyrazole and anthracene, and mono (N(CH2)2N(CH3)2) acridinone, have been compared with their respective aliphatic amine N-oxides -N(CH2)2N+(O-)(CH3)2. The results show that, unlike the intercalators, the N-oxides do not bind to DNA. Molecular modelling illustrates that the delta + nature of the intercalator alkylamino side chains in the protonated form allows for an attractive electrostatic interaction with phosphates of the DNA backbone, whereas the delta- partial charge on the N-oxide makes such an interaction not permissible; indeed, the electrostatic interaction with the DNA phosphates will be repulsive. The N-oxides show little or no cytotoxicity against V79 cells at concentrations equimolar to the IC90 (concentration that inhibits 90% of cell proliferation) of the respective intercalators. However, the cytotoxicity of anthrapyrazole N-oxide against hypoxic V79 cells in the presence of an activating system of S9 liver fraction was enhanced significantly. The results indicate that N-oxides of DNA-affinic agents have potential as bioreductive prodrugs, since they possess low aerobic toxicity but under hypoxic conditions can be metabolised to a potent cytotoxic species presumed to be a DNA-binding tertiary amine.

A comparison of free radical formation by quinone antitumour agents in MCF-7 cells and the role of NAD(P)H (quinone-acceptor) oxidoreductase (DT-diaphorase).

Electron paramagnetic resonance (EPR/ESR) spin trapping studies with DMPO revealed that purified rat liver NAD(P)H (quinone-acceptor) oxidoreductase (QAO) mediated hydroxyl radical formation by a diverse range of quinone-based antitumour agents. However, when MCF-7 S9 cell fraction was the source of QAO, EPR studies distinguished four different interactions by these agents and QAO with respect to hydroxyl radical formation: (i) hydroxyl radical formation by diaziquone (AZQ), menadione, 1AQ; 1,5AQ and 1,8AQ was mediated entirely or partially by QAO in MCF-7 S9 fraction; (ii) hydroxyl radical formation by daunorubicin and Adriamycin was not mediated by QAO in MCF-7 S9 fraction; (iii) hydroxyl radical formation by mitomycin C was stimulated in MCF-7 S9 fraction when QAO was inhibited by dicumarol; (iv) no hydroxyl radical formation was detected for 1,4AQ or mitoxantrone in MCF-7 S9 fraction. This study shows that purified rat liver QAO can mediate hydroxyl radical formation by a variety of diverse quinone antitumour agents. However, QAO did not necessarily contribute to hydroxyl radical formation by these agents in MCF-7 S9 fraction and in the case of mitomycin C, QAO played a protective role against hydroxyl radical formation.

What our enemies are really after: the other 90 percent.

As statistics have shown, physician income accounts for approximately 10 cents of the "health care dollar." But in this editorial, a Society trustee questions whether the real issue is not that 10 cents, but rather control of the other 90.

Tibiocalcaneal arthrodesis of a failed ankle fusion.

When previous attempts at ankle fusion have led to nonunion and destruction of both the talar body and its associated joint surfaces, treatment alternatives are limited. A case is presented where this type of pathology was addressed with a method of tibiocalcaneal arthrodesis that is rarely reported. This salvage procedure was completely successful in relieving the patient's symptoms in over 8 years of follow-up. The procedure and surgical alternatives are discussed in detail.

Reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells. II. Enhancement of the alkylating activity of AZQ by NAD(P)H: quinone-acceptor oxidoreductase (DT-diaphorase).

The alkylating activity of reduced diaziquone was studied by the nitrobenzylpyridine (NBP) assay and was compared to those of the parent compound and aziridine-containing N,N',N"-triethylenethiophosphoramide (Thio-TEPA). Diaziquone (AZQ) was reduced enzymatically by 2e- using S9 cell fraction from MCF-7 cells which is rich in NAA(P)H:quinone-acceptor oxidoreductase (DT-diaphorase) (QAO) activity. One electron enzymatic reduction was performed with NADPH-cytochrome c reductase. The alkylating activity of AZQ increased 3-fold when reduced by 2e-. This increase was inhibited by dicumarol, an inhibitor of QAO. In contrast, the alkylating activity of AZQ did not increase beyond that of the parent compound when reduced by 1e- using purified NADPH-cytochrome c reductase. Similar results were obtained when AZQ was reduced chemically with borohydride (2e-) and with NADPH (1e-). Anaerobic incubations of AZQ with the S9 fraction of MCF-7 cells (2e- reduction) resulted in an increase in NBP alkylation over its aerobic counterpart (1.8-fold) while maintaining the near 3-fold increase in alkylation over untreated AZQ. In contrast, AZQ incubations with NADPH-cytochrome c reductase (1e- reduction) under the same conditions did not result in an NBP alkylation increase over untreated AZQ. These results indicate that AZQ hydroquinone is most likely the responsible species for the observed alkylation of this antitumor agent to DNA and other nucleophiles. The results also suggest that NAD(P)H:quinone-acceptor oxidoreductase is a very important enzyme in the bioactivation of AZQ.

NAD(P)H (quinone acceptor) oxidoreductase (DT-diaphorase)-mediated two-electron reduction of anthraquinone-based antitumour agents and generation of hydroxyl radicals.

The anthraquinone-based antitumour agents mitoxantrone, daunorubicin and ametantrone were found to be substrates for NAD(P)H (quinone acceptor) oxidoreductase (DT-diaphorase) [QAO] isolated from rat liver. This was indicated by the stimulation of QAO-dependent NADPH oxidation by these agents. This effect followed Michaelis-Menten kinetics and was dependent on the concentration of QAO, inhibited by the specific QAO inhibitor dicumarol (15 microM) and enhanced by the QAO activators bovine serum albumin (0.01%) and Triton X-100 (0.03%). As indicated by the Vmax/Km ratio, mitoxantrone (26.53) was considerably more active than ametantrone (11.25) or daunorubicin (7.35). Metabolism of these anthraquinones was associated with the formation of superoxide anions, hydrogen peroxide and hydroxyl radicals as indicated by electron spin resonance spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide. This is likely to be due to the slow auto-oxidation of the respective dihydroquinones in the presence of molecular oxygen. QAO needs to be considered as a possible route of bioreductive activation of these agents.

DNA strand scission and free radical production in menadione-treated cells. Correlation with cytotoxicity and role of NADPH quinone acceptor oxidoreductase.

Menadione (MD; 2-methyl-1,4-naphthoquinone), a redox cycling quinone was shown to induce single (ss)- and double (ds)-strand DNA breaks in human MCF-7 cells. This DNA damage was mediated via the hydroxyl radical as evidenced by electron spin resonance spectroscopy (ESR) studies utilizing the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide. The free radical production and DNA damage were shown to play a role in MD cytotoxicity as revealed by the reversal of MD toxicity and inhibition of hydroxyl radical production by exogenously added catalase. The role of NADPH quinone acceptor oxidoreductase in the metabolism of MD was evaluated. Purified quinone acceptor oxidoreductase in combination with MD resulted in the production of significant levels of the hydroxyl radical as measured by ESR. Dicumarol, an inhibitor of quinone acceptor oxidoreductase, decreased the production of the hydroxyl radical and attenuated DNA strand breaks in MCF-7 cells treated with MD.

Lack of involvement of reactive oxygen in the cytotoxicity of mitoxantrone, CI941 and ametantrone in MCF-7 cells: comparison with doxorubicin.

The MCF-7 cell S9 fraction and whole MCF-7 cells can mediate one-electron-redox cycling of doxorubicin, giving rise to concomitant oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), formation of a drug semiquinone free radical, consumption of molecular oxygen and formation of superoxide anions and hydroxyl radicals. Doxorubicin redox cycling was consistent with DNA strand breakage and cell kill in MCF-7 cells. In contrast, no evidence for redox cycling was found for mitoxantrone (MIT), CI941 or ametantrone (AMET) in MCF-7 cells. Despite the absence of redox cycling, the CI941, MIT, and AMET concentrations resulting in 50% mortality (LC50; 1.5 x 10(-10), 5.2 x 10(-9) and 1.2 x 10(-6) M, respectively) of MCF-7 cells were lower than that of DOX (3.0 x 10(-6) M). Furthermore, the higher cytotoxicity of MIT and CI941 as compared with AMET or DOX was associated with greater efficiency in inducing DNA strand breakage in MCF-7 cells as determined by alkaline elution. Since MIT and CI941 proved to be the most potent DNA-damaging and cytotoxic agents in this study, the ability of DOX to undergo redox cycling does not appear to confer increased cytotoxic potential on this agent. The present study revealed several important aspects with regards to the structural modification of anthraquinone antitumour agents. Firstly, the C1 and C4 positioning of the hydroxyethylamino side chains on MIT, CI941 and AMET is associated with a lack of flavin reductase-mediated activation of these agents. Secondly, the possession of a C5 or C8 aromatic hydroxyl group appears to be intimately involved in the enhanced DNA strand breakage and cytotoxic potency of MIT and CI941, since AMET does not possess these groups. These findings indicate that future development of quinone antitumour agents should concentrate on compounds that do not undergo redox cycling but do possess aromatic hydroxyl groups, since the latter appear to be responsible for the enhanced cytotoxicity of MIT and CI941.

The reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells: free radical formation and NAD(P)H: quinone-acceptor oxidoreductase (DT-diaphorase) activity.

The S9 fraction of MCF-7 human breast carcinoma cells has NAD(P)H (quinone-acceptor) oxidoreductase activity as measured by the reduction of dichlorophenol-indophenol (DCPIP). This reduction is dependent on the activators Tween-20 and bovine serum albumin and it is inhibitable by dicumarol. The S9 fraction also has cytochrome c reductase activity which is approximately 29 times less than the two-electron reduction activity of NAD(P)H (quinone-acceptor) oxidoreductase. Diaziquone (AZQ) is a substrate for this NAD(P)H oxidoreductase active S9 fraction as judged by its enzymatic reduction detected spectrophotometrically and by electron spin resonance spectroscopy. Two-electron mediated enzymatic reduction of AZQ was evidenced by the formation of the colorless dihydroquinone (AZQH2) which could be followed at 340 nm. The production of the dihydroquinone was inhibitable by dicumarol implicating NAD(P)H oxidoreductase in its formation. Under aerobic conditions, electron spin resonance spectroscopy showed evidence for the production of AZQ semiquinone (AZQH) and oxygen radicals. Under anaerobic conditions no oxygen radicals were observed, but the semiquinone was stable for hours. These results are also inhibitable by dicumarol and suggest a two-step one-electron oxidation process of the dihydroquinone. The production of semiquinone and oxygen radicals as detected by electron spin resonance spectroscopy was more sensitive to dicumarol when NADPH was used as cofactor (68% inhibition of OH and 65% inhibition of AZQH) than when NADH was used (28% inhibition of OH and 5% inhibition of AZQH). This suggests that NADH flavin reductases play a more important role in the one-electron reduction pathway of AZQ in MCF-7 S9 fraction than NADPH reductases. The reduction of AZQ by NAD(P)H (quinone-acceptor) oxidoreductase may play an important role in the bioreductive alkylating properties of AZQ.

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.