With NADPH as the electron donor, rat liver NADPH cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) catalyzes the single-electron reduction of several quinone antibiotics to a semiquinone or free radical state. The benzanthraquinones adriamycin, daunorubicin, carminomycin, 7-O-methylnogalarol, and aclacinomycin A and the N-heterocyclic quinones streptonigrin and mitomycin C are activated to free radical intermediates which can transfer their single electron to molecular oxygen to form superoxide. The overall Km range for this electron transfer is 0.4 to 42.1 X 10-4 M. We postulate that the formation of the "site-specific free radical" intermediate is central to the cytotoxic action of these antibiotics.An unusually high proportion of anticancer agents contain quinone groups. Such quinone-containing anticancer agents include adriamycin, daunorubicin, mitomycin C, streptonigrin, lapachol, and analogs of these agents. Although investigators have identified several potential biochemical sites of action for these drugs, preponderant evidence indicates that the agents act primarily by interfering with DNA and RNA replication(1-4).We have established that these quinone agents are catalytically activated to a free radical state by a microsomal system requiring NADPH as an electron donor (5, 6). Normal microsomes as well as microsomes from murine leukemia cells catalyze augmented oxygen consumption with the quinone antibiotics, indicative of free radical formation. By using effectors of this microsomal system we obtained indirect evidence that a flavoprotein catalyst of the microsomes was involved and that cytochrome P-450 was not. Iyanagi and Yamazaki (7) have shown that NADPH cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) reduces quinone substrates to semiquinones; and Goodman and Hochstein (8) reported that NADPH cytochrome P-450 reductase catalyzes production of superoxide from adriamycin. In the present paper, we describe and characterize the catalysis of drug free-radical formation bhey -tochrome P-450 reductase from rat-iver microsomes and propose a mechanism for the cytotoxic action of these agents.METHODS AND MATERIALS Adriamycin, N-dimethyladriamycin, daunorubicin, N-acetyldaunorubicin, 7-iminodaunorubicin, aclacinomycin A, mitomycin C, streptonigrin, and lapachol were kindly provided by the Drug Synthesis and Chemistry Branch and the Natural Products Branch (Division of Cancer Treatment, National Cancer Institute, National Institutes of Health). Daunorubicinol (9) and adriamycin (Fe3+)4 (10) were prepared as described. Carminomycin was provided by G. F. Gause (Institute of New Antibiotics, Moscow). Rubidazone was supplied by Rene Maral (Rhone-Poulenc, Vitry-Sur-Seine, France). Nogalamycin, 7-O-methylnogarol, and steffimycin were provided by G. Neil (Upjohn Co.); and N-trifluoroacetyladriamycin-14-valerate (AD-32) and N-trifluoroacetyladriamycin (AD-41) were supplied by M. Israel, (Farber Cancer Center, Boston, MA).NADPH cytochrome P-450 reduct...

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The effects of PTH on phosphate fluxes and cAMP and protein secretion in rat parotid cell aggregates were studied. PTH-(1-34) as well as PTH-(1-84) stimulated phosphate efflux in a dose-dependent [5 X 10(-7) to 7 X 10(-6) M for human (h) PTH-(1-34), 2 X 10(-7) to 5 X 10(-6) M for bovine (b) PTH-(1-34), and 3.8-114 U/ml for bPTH-(1-84)] and time-dependent [detectable by 15-30 min after incubation with N-terminal PTH and 30-45 min after incubation with bPTH-(1-84)] manner. The effect of PTH on phosphate efflux was saturable, with maximal stimulation at 10(-6) M above for N-terminal PTH and at 57.8 U/ml and above for bPTH-(1-84). Half-maximal stimulation of phosphate efflux by hPTH-(1-34), bPTH-(1-34), and bPTH-(1-84) was achieved at 5.7 X 10(-7) M, 10(-7) M, and 40 U/ml, respectively. Neither bPTH-(1-34) nor bPTH-(1-84) had any effect on phosphate uptake by parotid cell aggregates at the concentrations examined. bPTH-(1-34) and bPTH-(1-84) also stimulated amylase secretion, with a significant increase in cAMP production (70-90% over basal). However, (BU)2cAMP did not stimulate phosphate efflux. Isoproterenol (10(-5) M) markedly stimulated cAMP production and amylase secretion without causing a significant increase in phosphate efflux. Oxidized bPTH-(1-84) and the C-terminal fragment of hPTH had no stimulating effect on phosphate efflux. The present results suggest, therefore, that PTH may play an important role in regulating rat parotid cell phosphate metabolism through an apparently cAMP-independent process.