tirapazamine and Carcinoma-256--Walker

As judged by alkaline elution, exposure of Walker cells to either 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) or nitromin results in a dose-dependent increase in DNA damage due to single-strand breaks. With nitromin or SR 4233 there was little difference in the extent of DNA single-strand breaks between Walker cells incubated either hypoxically or aerobically. In contrast, there was a 24-fold enhancement in the differential hypoxic/aerobic response to SR 4233 in clonogenic studies. Following incubation of cells with nitrogen mustard, DNA cross-linking is observed. Bioreduction of nitromin would be expected to yield nitrogen mustard as the putative reactive metabolite. However, only DNA strand-breaks could be detected in Walker cells incubated with nitromin, suggesting that reduction of this pro-drug to nitrogen mustard was not a major activation pathway. In cells incubated under aerobic conditions, SR 4233 induces oxidative DNA damage, as indicated by the formation of 8-hydroxydeoxyguanosine, suggesting the involvement of futile redox cycling. In rats dosed with SR 4233 in vivo, significantly higher levels of 8-hydroxydeoxyguanosine could be detected in liver, compared to vehicle-dosed controls.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aerobiosis; Animals; Antineoplastic Agents; Biotransformation; Carcinoma 256, Walker; Cell Hypoxia; Deoxyguanosine; DNA Damage; DNA, Single-Stranded; Liver; Mechlorethamine; Oxidation-Reduction; Rats; Tirapazamine; Triazines; Tumor Cells, Cultured

DT-diaphorase is a unique two electron (2e) donating reductase catalyzing either bioactivation or bioprotection reactions. Using human and rodent DT-diaphorase preparations (cell extracts and purified enzyme) we have characterized the reductive metabolism of the hypoxic cell cytotoxins EO9, mitomycin C (MMC), CB 1954, and SR 4233 in vitro. Drug metabolism was assayed spectrophotometrically or by HPLC, with dicoumarol as a selective inhibitor. DNA damage was measured using an agarose gel mobility technique with plasmid pBR322 DNA. The developmental indoloquinone, EO9, was metabolized by both rat Walker and human HT29 tumor DT-diaphorases. Reduction proceeded 5-fold more efficiently with the rat than the human tumor enzyme and resulted in single-strand breaks in plasmid DNA. The structurally related MMC was metabolized much more slowly than EO9 by the rat Walker tumor enzyme and there was no detectable reaction with the human HT29 tumor DT-diaphorase. No DNA damage was seen with MMC for either enzyme. The dinitrophenylaziridine CB 1954 was reduced by both human and rat enzymes forming, preferentially, the highly toxic 4-hydroxylamine as a 4e reduction product. Rates were 3-fold lower than for the human tumor enzyme. SR 4233 was also reduced by the rat tumor enzyme predominantly via 4e reduction to the benzotriazine SR 4330, in a novel reaction mechanism. This appears to be a bioprotection pathway that bypasses the toxic 1e radical formed by other reductases. Such information may be valuable in the selection of hypoxic cell cytoxins to treat human tumors high or low in DT-diaphorase and should facilitate 'enzyme-directed' analogue development.

Topics: Animals; Antineoplastic Agents; Aziridines; Carcinoma 256, Walker; Cell Hypoxia; Colonic Neoplasms; Humans; In Vitro Techniques; Indolequinones; Indoles; Mitomycin; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Prodrugs; Rats; Tirapazamine; Triazines

3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233; WIN 59075) is a highly selective hypoxic cell cytotoxin soon to enter phase I clinical trial. The compound is thought to exert its action through a toxic one-electron reduced free radical intermediate. Preliminary data have suggested that SR 4233 may be metabolized by DT-diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2)] to both two- and four-electron reduced products and that this route of biotransformation may represent a bioprotection pathway. In this study, a highly purified enzyme preparation was employed in order to investigate further the metabolism of SR 4233 by DT-diaphorase and to examine the mechanism of reduction in more detail. Spectrophotometric analysis showed that SR 4233 underwent reduction by DT-diaphorase with an apparent Km of 1.23 +/- 0.27 mM and Vmax of 8.55 +/- 1.67 nmol/min/microgram protein. This reaction was inhibited completely by dicoumarol (100 microM) and partially by an antiserum raised against the purified enzyme. Characterization of the products of SR 4233 reduction by reverse-phase HPLC confirmed that both two- (SR 4317) and four- (SR 4330) electron reduction products were generated, the latter being the predominant metabolite, particularly in prolonged incubations. Further experiments showed that the four-electron reduction product, but not the two-electron reduction product, was also a substrate for DT-diaphorase with an apparent Km of 1.14 mM and a Vmax of 57.12 nmol/min/micrograms protein. The results presented confirm that SR 4233 is indeed a substrate for DT-diaphorase and that a mixture of two-, four- and six-electron reduced products may be formed. The possible toxicological and pharmacodynamic significance of this metabolism is discussed.

Topics: Animals; Carcinoma 256, Walker; Cell Hypoxia; Cell Line; Chromatography, High Pressure Liquid; Kinetics; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Rats; Tirapazamine; Triazines

Topics: Aerobiosis; Anaerobiosis; Animals; Antineoplastic Agents; Carcinoma 256, Walker; Cell Line; Cell Transformation, Neoplastic; DNA Damage; DNA Replication; Kinetics; Mechlorethamine; Mice; Nitrogen Mustard Compounds; Rats; Thymidine; Tirapazamine; Triazines