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Biomedical subjects

R F Novak

Publications and source records attributed to R F Novak.

At least 91 records · Page 5Linked to original sources
PubMed1984

Spectroscopic evidence for anthracenedione antineoplastic agent self-association and complex formation with flavin nucleotides.

Dihydroxyanthraquinone (DHAQ) and ametantrone (anthraquinone) are two new anthracenedione antineoplastic agents which were found by proton NMR spectroscopy to self-associate in aqueous media. Self-association was consistent with a bimolecular model, with average association constant values of 3400 and 2900 M-1 determined for DHAQ and ametantrone, respectively. Both anthracenediones interacted with the flavin nucleotides FMN and FAD to produce concentration-dependent upfield shifts of the flavin isoalloxazine ring proton signals, as observed by proton NMR spectroscopy. Average association constant values obtained for FMN-DHAQ, FAD-DHAQ, FMN-ametantrone, and FAD-ametantrone complexation were 5100, 2600, 4300, and 1600 m-1, respectively. Optical difference spectroscopy confirmed FMN-DHAQ complexation, which resulted in a hyperchromic, bathochromic shift of the DHAQ spectrum following addition of FMN. These results were consistent with the formation of a pi-pi bimolecular ring-stacking complex. Information obtained on anthracenedione self-association and complexation with flavins may be of consequence in the interpretation of anthracenedione-DNA binding data and flavoprotein-mediated anthracenedione metabolic activation.

Anthraquinones↗
PubMed1984

Induction of rabbit hepatic microsomal cytochrome P-450 by imidazole: enhanced metabolic activity and altered substrate specificity.

Pretreatment of rabbits with imidazole resulted in a twofold increase in hepatic microsomal cytochrome P-450 content, with the apparent induction of two or more distinct forms of the cytochrome [K. K. Hajek and R. F. Novak (1982) Biochem. Biophys. Res. Commun. 108, 664-672]. The metabolic properties of imidazole-induced microsomes have been compared to those of uninduced, phenobarbital- and beta-naphthoflavone-induced preparations. Metabolic activity was enhanced as a consequence of increased P-450 content and as a result of the presence of different forms of the cytochrome. When rates were expressed per nanomole P-450 the following were observed: (a) p-nitroanisole O-demethylation was comparable in all preparations; (b) N,N-dimethylaniline N-demethylation was comparable in imidazole- and beta-naphthoflavone-induced, and uninduced microsomes; (c) polycyclic aromatic hydrocarbon hydroxylase activity was approximately twofold greater in imidazole-induced relative to phenobarbital-induced microsomes, but was only one-half that of beta-naphthoflavone-induced microsomes; and (d) metabolism of N,N-dimethylnitrosamine was enhanced fivefold, alcohol oxidation increased three- to fivefold, and aniline hydroxylation was threefold greater in imidazole-induced microsomes compared to phenobarbital- or beta-naphthoflavone-induced preparations. Eadie-Scatchard analysis yielded a single Km value for dimethylnitrosamine N-demethylase activity in imidazole-induced microsomes; in contrast, both high- and low-Km values were obtained for phenobarbital- or beta-naphthoflavone-induced microsomal preparations. Dimethylnitrosamine N-demethylase activity was P-450 dependent; neither flavin monooxygenase nor monoamine oxidase appeared to contribute significantly to dimethylnitrosamine metabolism. Dimethyl sulfoxide was a competitive inhibitor of dimethylnitrosamine N-demethylase activity in imidazole-, phenobarbital-, and beta-naphthoflavone-induced microsomes. Dimethyl sulfoxide competitively inhibited ethanol oxidation in imidazole-induced microsomes; it was a noncompetitive inhibitor of ethanol oxidation in phenobarbital- or beta-naphthoflavone-induced microsomes.

Alcohols↗
PubMed1984

Ametantrone inhibits prostaglandin--mediated resorption in bone organ culture.

Prostaglandins (PG) have been postulated to be involved in both tumor metastases to bone and in tumor-induced bone resorption. The anthracenedione antineoplastic agents ametantrone (HAQ) and mitoxantrone are potent antioxidants and inhibit hydroperoxide-dependent initiation and propagation reactions. Therefore, these compounds may inhibit PG production and could also inhibit tumor metastases and tumor-induced resorption. The ability of HAQ, a prototypic anthracenedione, to inhibit PG synthesis and PG-mediated bone resorption was investigated using neonatal mouse calvaria in organ culture. Epidermal growth factor (EGF) stimulates bone resorption in this tissue by inducing PG synthesis. Consequently, if HAQ inhibits EGF-stimulated PG synthesis, it should also inhibit EGF-stimulated bone resorption. HAQ, at 10 microM, completely abolished EGF-stimulated PG synthesis and calcium release. Moreover, HAQ (1.0-30 microM) inhibition of EGF-stimulated PGE2 synthesis correlated with the inhibition of EGF-stimulated Ca release in a concentration-dependent manner. In contrast to EGF, parathyroid hormone stimulates resorption by a PG-independent pathway. HAQ at 10 microM had no effect on parathyroid hormone stimulated Ca release. These results suggest that HAQ inhibition of bone resorption appears to be primarily mediated by inhibition of PG biosynthesis.

Animals↗
PubMed1983

Bis(alkylamino)anthracenedione antineoplastic agent metabolic activation by NADPH-cytochrome P-450 reductase and NADH dehydrogenase: diminished activity relative to anthracyclines.

Stimulation of the rates of NAD(P)H oxidation, superoxide generation, and hydrogen peroxide formation by three anthracenedione antineoplastic agents in the presence of NADPH-cytochrome P-450 reductase, NADH dehydrogenase, or rabbit hepatic microsomes was studied and the results compared with those obtained for the anthracyclines Adriamycin and daunorubicin. In all cases the anthracenediones, including mitoxantrone and ametantrone, were significantly (5- to 20-fold) less effective than the anthracyclines in stimulating NAD(P)H oxidation, superoxide formation, or hydrogen peroxide production. Of the three anthracenediones studied, the ring-monohydroxylated compound showed the greatest activity followed by the ring-dihydroxylated derivative (mitoxantrone). In contrast, the non-ring-hydroxylated anthracenedione (ametantrone) was a relatively ineffective electron acceptor and inhibited the reduction of more effective acceptors such as Adriamycin. Michaelis-Menten kinetic constants were determined by analysis of the rates of NADPH oxidation. NADP+ and 2'-AMP inhibited the reduction of the ring-hydroxylated anthracenediones and anthracyclines, demonstrating the enzymatic nature of the reaction. The non-ring-hydroxylated anthracenedione inhibited the reduction of Adriamycin by both P-450 reductase and NADH dehydrogenase with 50% inhibition achieved at approximately 300 microM. Thus, there appears to exist a structural relationship between anthracenedione ring hydroxylation and metabolic activation. These results also suggest that the relative inability of the anthracenediones to function as artificial electron acceptors in comparison to the anthracyclines may be correlated with diminished anthracenedione cardiotoxicity.

Animals↗
PubMed1983

Inhibitory effects of anthracenedione antineoplastic agents on hepatic and cardiac lipid peroxidation.

The effects of mitoxantrone, ametantrone and a monohydroxylated anthracenedione on hepatic microsomal, cardiac sarcosomal and cardiac mitochondrial lipid peroxidation were examined and compared with those of doxorubicin and daunorubicin. Rabbit microsomal NADPH-dependent lipid peroxidation was inhibited by the anthracenediones in a concentration-dependent manner, whereas doxorubicin caused a concentration-dependent enhancement of peroxidation. Mitoxantrone and ametantrone (200 microM) completely inhibited microsomal malondialdehyde production while an identical concentration of doxorubicin caused a 2.5-fold stimulation. Rabbit cardiac sarcosomal NADPH-dependent malondialdehyde production was also abolished by 100 microM anthracenedione. Mitochondria isolated from rabbit hearts were found to support NADH-dependent lipid peroxidation. Doxorubicin produced a maximal 3-fold enhancement of mitochondrial malondialdehyde production at 25 microM. The anthracenediones however, completely inhibited mitochondrial lipid peroxidation Drug-stimulated lipid peroxidation was also effectively diminished by mitoxantrone and ametantrone in a concentration-dependent manner. Half-maximal inhibition of doxorubicin-stimulated rabbit microsomal malondialdehyde production was achieved by 4 anal 6 microM mitoxantrone and ametantrone, respectively. Furthermore this effect was not limited to anthracycline-induced lipid peroxidation. Mitoxantrone and ametantrone also protected against rat microsomal lipid peroxidation produced by nitrofurantoin, paraquat and doxorubicin, decreasing these rates by 80, 90, and 50%, respectively, at 10 microM anthracenedione. The relative inability of the anthracenediones to stimulate lipid peroxidation is consistent with the diminished cardiotoxicity of ametantrone and mitoxantrone relative to doxorubicin and daunorubicin.

Animals↗
PubMed1982

Generation of superoxide via the interaction of nitrofurantoin with oxyhemoglobin.

Nitrofurantoin was found to interact with HbO2 to cause the concomitant formation of methemoglobin and superoxide. The rate of formation of methemoglobin and superoxide was linearly dependent upon the concentration of nitrofurantoin and could be inhibited by superoxide dismutase, catalase, or the prior conversion of HbO2 to ethylioscyanoferrohemoglobin. The ability of nitrofurantoin to interact with HbO2 and cause superoxide formation may represent one mechanism by which it produces red cell toxicity and suggests that generation of superoxide in erythrocytes may occur via a different mechanism than that which occurs in microsomes.

Animals↗
PubMed1982

The acute vasculitis of Wegener's granulomatosis in renal biopsies.

The kidney biopsy specimens from five patients with Wegener's Granulomatosis were reviewed in an attempt to characterize the early histological lesion when vasculitis was present. The vascular lesions were found mainly in interlobular-sized arteries. The acute vascular lesions were sparse and focal, mainly localized to the intimal area, and with fibrinoid material and platelets constituting the early infiltrate. The electron microscopy and scanning electron microscopy of the involved areas further emphasized the presence of platelets in the early lesion, and accentuated endothelial alterations occurring along with the intimal infiltrate. Morphologic evidence of immune complex presence was not found.

Adult↗
PubMed1982

Studies on the mechanism of nitrofurantoin-mediated red cell toxicity.

The mechanism of nitrofurantoin-mediated depletion of red cell reduced glutathione (GSH) was investigated. Nitrofurantoin caused cellular depletion of GSH in vitro under aerobic and oxygen-depleted conditions, an effect which could be partially inhibited by coincubation with the hemeprotein ligand ethyl isocyanide, or completely prevented by coincubation with 2'-AMP, an inhibitor of NADPH-dependent reductase enzymes. Covalent binding of nitrofurantoin to red cell macromolecules appeared to be a minor process and was not substantially inhibited by either ethyl isocyanide or 2'-AMP. Covalent binding was only slightly greater under oxygen-depleted conditions. Nitrofurantoin increased the rate of superoxide formation in red cell lysate, an effect inhibited by ethyl isocyanide but not by 2'-AMP. These data suggest different mechanisms for nitrofurantoin-mediated depletion of GSH under aerobic and oxygen-depleted conditions. In the presence of oxygen, nitrofurantoin causes the release of superoxide from oxyhemoglobin. The superoxide thus formed may deplete GSH via several mechanisms. In the absence of oxygen, nitrofurantoin is reduced to reactive metabolites via reactions which appear to require the participation of both an NADPH-dependent flavoprotein and hemoglobin.

Adenosine Monophosphate↗
PubMed1982

Inhibition of microsomal oxidative drug metabolism by 1,4-bis (2-[(2-hydroxyethyl)amino]-ethylamino)-9,10-anthracenedione diacetate, a new antineoplastic agent.

The effects of 1,4-bis(2-[(2-hydroxyethyl)amino]-ethylamino)-9,10-anthracenedione diacetate (HAQ) on rabbit liver microsomal oxidative drug metabolism were investigated. HAQ was found to inhibit O-dealkylase and N-demethylase activities in phenobarbital-induced microsomes, and aryl hydrocarbon hydroxylase activity in beta-naphthoflavone-induced microsomes. The inhibition was noncompetitive with respect to substrate concentration, with inhibitory constant (Ki) values of 2.9, 2.6, and 3.0 mM for p-nitroanisole, N,N-dimethylaniline, and benzo[a]pyrene, respectively. In contrast, HAQ failed to inhibit p-nitroanisole metabolism when the reaction was supported with cumene hydroperoxide. HAQ also inhibited basal and substrate-stimulated microsomal NADPH oxidation. The degree of inhibition of NADPH oxidation and product formation were comparable. These data, in conjunction with the results of previous studies, suggest that HAQ inhibits electron transfer by microsomal NADPH-cytochrome P-450 reductase, diminishing electron flow to cytochrome P-450 and thereby inhibiting substrate metabolism. This mechanism differs markedly from that for inhibition of drug metabolism by other quinones, such as menadione, in which accelerated electron flow through P-450 reductase to the quinone diverts reducing equivalents from cytochrome P-450.

Animals↗
PubMed1980

N6-Trimethyl-lysine metabolism. 3-Hydroxy-N6-trimethyl-lysine and carnitine biosynthesis.

Rats injected with N6-[Me-3H]trimethyl-lysine excrete in the urine five radioactively labelled metabolites. Two of these identified metabolites are carnitine and 4-trimethylammoniobutyrate. A third metabolite, identified as 5-trimethylammoniopentanoate, is not an intermediate in the biosynthesis of carnitine; the fourth and major metabolite, N2-acetyl-N6-trimethyl-lysine, is not a precursor of carnitine. The remaining metabolite (3-hydroxy-N6-trimethyl-lysine) is converted into trimethylammoniobutyrate and carnitine by rat liver slices and into trimethylammoniobutyrate by rat kidney slices. In rat liver and kidney-slice experiments, radioactivity from DL-N6-trimethyl-[1-14C]lysine and DL-N6-trimethyl-[2-14C]lysine was incorporated into N2-acetyl-N6-trimethyl-lysine and 3-hydroxy-N6-trimethyl-lysine, but not into trimethylammoniobutyrate or carnitine. A procedure was devised to purify milligram quantities of 3-hydroxy-N6-trimethyl-lysine from the urine of rats injected chronically with N6-trimethyl-lysine (100 mg/kg body wt. per day). The structure of 3-hydroxy-N6-trimethyl-lysine was confirmed chemically and by nuclear-magnetic-resonance spectrometry [Novak, Swift & Hoppel (1980) Biochem. J. 188, 521--527]. The sequence for carnitine biosynthesis in liver is: N6-trimethyl-lysine leads to 3-hydryxy-N6-trimethyl-lysine leads to leads to 4-trimethylammoniobutyrate leads to carnitine.

Animals↗
PubMed1980

N6-Trimethyl-lysine metabolism. Structural identification of the metabolite 3-hydroxy-N6-trimethyl-lysine.

(1)H and (13)C nuclear-magnetic-resonance spectroscopy and functional-group analysis were used to determine the molecular structure of an isolated metabolite (II(b)) of trimethyl-lysine as 3-hydroxy-N(6)-trimethyl-lysine, an important intermediate in the conversion of trimethyl-lysine into trimethylammoniobutyrate and carnitine [Hoppel, Cox & Novak (1980) Biochem. J.188, 509-519]. Functional-group analysis revealed the presence of a primary amine and reaction of metabolite (II(b)) with periodate yielded 4-N-trimethylammoniobutyrate as a product, showing 2,3-substitution on the molecule and suggesting that the 3-substitution on the molecule may be an alcohol ([unk]CH-OH), amine ([unk]CH[unk]-NH(2)) or carbonyl ([unk]C=O) functional group. (1)H integration ratios, (1)H and (13)C chemical-shift data and (1)H and (13)C signal multiplicities from the sample (II(b)) were used to complete the identification of metabolite (II(b)) as 3-hydroxy-N(6)-trimethyl-lysine. For example, the proton multiplet at delta 4.2p.p.m. and doublet at delta 4.1p.p.m., positions representative of amine or alcohol substitution on methylene carbon atoms, integration ratios of 1:1:2:9:4 and a positive ninhydrin test suggest 3-hydroxy-N(6)-trimethyl-lysine as the molecular structure for metabolite (II(b)). (13)C chemical-shift data obtained from the sample (II(b)) and compared with several model compounds (trimethylammoniohexanoate, trimethyl-lysine and 3-hydroxylysine) resulted in generation of the spectrum of the metabolite and allowed independent identification of metabolite (II(b)) as 3-hydroxy-N(6)-trimethyl-lysine. The (1)H spectrum of erythro- and threo-3-hydroxylysine are presented for comparison, and the (1)H and (13)C n.m.r. spectra of the erythro-isomer support this analysis.

Carbon Radioisotopes↗