Mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates

Summary.-Twenty-one nitroheterocycles, including metronidazole, misonidazole and AF-2, were tested for their ability to inhibit DNA synthesis in mouse L-929 cells growing in culture. All those tested inhibited the rate of incorporation of 3H -thymidine into L cells following drug treatment for 4 h under aerobic conditions. Only 4 drugs reached their limits of solubility before the uptake of 3H-thymidine was inhibited by 50%o or more. For the remaining 17, the log of the concentration producing 50%o inhibition of incorporation was directly correlated with the half-wave reduction potential of the compound.

Section: Resultsmentioning

confidence: 99%

Inhibition of DNA synthesis by nitroheterocycles I. Correlation with half-wave reduction potential

Olive¹

1979

“…The metabolism of RSU1069 has not been studied; however, for 2-nitroimidazoles generally, benznidazole and misonidazole have been used as model drugs (Mason and Holtzman, 1975;Walton and Workman, 1987). Using rat liver microsomes it has been shown that, in hypoxia, P450 reductase is important for conversion of the nitro compound to the corresponding nitro radical anion, which is a prerequisite for downstream conversion to the nitroso and hydroxylamino analogues -thought to be the toxic, alkylating species (Noss et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

Overexpression of human NADPH:cytochrome c (P450) reductase confers enhanced sensitivity to both tirapazamine (SR 4233) and RSU 1069

Patterson

Saunders²,

Chinje³

et al. 1997

Summary P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine Br J Cancer 72: 1144-1150. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069.Keywords: P450 reductase; hypoxia, bioreductive drugs; tirapazamine; RSU 1069; breast cancer It is now well established that regions of low oxygen tension (hypoxia) can exist in many human solid tumours and that this hypoxia can sometimes predispose to failure of some treatments with radiotherapy (Hoeckel et al, 1996). One of the reasons for this failure is likely to be the inherent radiation resistance of hypoxic cells. One strategy being developed to overcome this problem is via the use of drugs that will selectively kill hypoxic cells Stratford, 1986, 1994;Zeman et al, 1986;Kennedy, 1987;Brown and Siim, 1996;Denny et al, 1996). This approach involves the exploitation of various biochemicla processes that, at low oxygen tensions, can result in selective reductive activation of a prodrug to give cytotoxic species (Workman and Stratford, 1993 enzymes. Thus, it has been proposed that hypoxic cells could be more effectively targeted if differences in the levels of the reductases in various cell types were taken into account to direct appropriate agents to particular human tumours based on their enzymology Workman and Stratford, 1993). Patterson et al (1995) examined the possible application of the enzyme-directed approach to the development of tirapazamine. The metabolism of this agent was suggested to be dependent on the presence of P450 reductase (Walton et al, , 1992. In a panel of six breast cancer cell lines with a sixfold range in P450 red...

“…It is interesting to speculate on the mechanism of this interaction. A previous report from this laboratory (Hall et al, 1977) suggested that both the radiosensitizing and cytotoxic properties of MIS on hypoxic cells are mediated via a common metabolite, the RN02-radical anion (Mason & Holtzman, 1975;Wardman & Clarke, 1976). When cysteamine, a free-radical scavenger, was added in equimolar concentrations with MIS both the radiosensitization and cytotoxicity of MIS were reversed.…”

Section: Fig 2 Continuation Ofmentioning

confidence: 96%

Misonidazole and MTDQ in combination: Cytotoxic and radiosensitizing properties in hypoxic mammalian cells

Astor¹,

Hall²

1979

Summary.-A combination of misonidazole and MTDQ (6,6'-methylene-bis-2,2,4 trimethyl-1,2-dihydroquinoline) has been tested for its radiation-sensitizing properties and cytotoxicity, using Chinese hamster V79 cells cultured in vitro.Both compounds sensitize hypoxic cells to the effects of X-rays, and when used in combination their sensitizing properties are additive. By contrast, the presence of MTDQ completely inhibits the cytotoxicity that misonidazole exhibits towards hypoxic cells.These experiments shed some light on the mechanism of action of electron-affinic hypoxic cell sensitizers, and the combination of radiosensitizers suggested may have an application in human cancer radiotherapy by eliminating the neurotoxicity experienced by patients receiving misonidazole during radiotherapy.