NAD(P)H nitroblue tetrazolium reductase levels in apparently normoxic tissues: a histochemical study correlating enzyme activity with binding of radiolabelled misonidazole

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Summary The tritium-labelled analogues of pimonidazole and RSU 1069 were injected into mice bearing the KHT murine sarcoma which has a hypoxic cell fraction of 1-0%. The distribution of activity at 24 h was recorded using autoradiography and measurement of tissue activity. Autoradiographs with both drugs showed high activity in particular cells within tumour, eye (melanin-associated cells), eyelid (Meibomian gland), liver (centrilobular area), skin (sebaceous gland and melanin), stomach (squamous area), footpad, oesophagus, labial gland, Zymbal's gland, preputial gland, parotid gland (intralobular ducts) and airway epithelium. These tissues had previously been identified as sites of binding of misonidazole. The measurement of total tissue radioactivity showed significantly higher activity in liver, eyelid (Meibomian gland), oesophageal lining, kidney and labial gland than was found in the tumour.Misonidazole (1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol; MISO) sensitises hypoxic tumour cells to ionising radiation (Asquith et al., 1974). This radiosensitisation is associated with the electron affinity of, MISO and is a fast (subsecond) free-radical process. MISO can also be involved in a separate, slower, enzyme dependent process in which, in the presence of the appropriate reductase(s), the nitro group is reduced yielding cytotoxic species (Varghese et al., 1976;Rauth, 1984). This second process is favoured by a hypoxic environment -such as occurs in parts of many tumoursand this leads to locally enhanced retention. The concept of the preferential retention of reactive bioreduced metabolites in tumours has generated two lines of research. One is directed towards tumour therapy (bioreductive drug therapy). In this, drugs are being developed which, as with MISO, on reduction in the hypoxic milieu of the tumour, form cytotoxic metabolites(s) that bind to critical macromolecules such as DNA. The other line of research is in the field of imaging and spectroscopy.

Section: Discussionmentioning

confidence: 63%

Section: Discussionmentioning

confidence: 70%

mentioning

confidence: 99%

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Distribution of pimonidazole and RSU 1069 in tumour and normal tissues

Cobb¹,

Nolan²,

Butler³

1990

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“…It has been reported that human gliomas contain bioreductive enzymes [NAD(P)H-cytochrome P450 oxidoreductase, NAD(P)H -quinone oxidoreductase] (Rampling et al, 1994), which might be involved in the reductive activation of nitroimidazoles (Cobb et al, 1990;Parliament et al, 1992;Joseph et al, 1994). Thus, the finding that unresected, untreated human malignant gliomas failed to show avidity for the hypoxia marker ['231]IAZA in situ was unexpected.…”

Section: Discussionmentioning

confidence: 99%

Anomalous patterns of nitroimidazole binding adjacent to necrosis in human glioma xenografts: possible role of decreased oxygen consumption

Parliament¹,

Franko²,

Allalunis-Turner³

et al. 1997

Summary In contrast to reports of extensive hypoxia in human gliomas in situ measured by P02 histography, non-invasive methods of assessing glioma oxygenation, including nitroimidazole binding, have yielded surprisingly contradictory results. In order to investigate the relationship of necrosis, hypoxia, nitroreductase activity and cellular respiration in human gliomas, subcutaneous models using the human glioma cell lines M059K, M006 and M01 Ob were developed in the murine SCID host. Intracranial growth of the M006 line was achieved in nude rats. The nitroreductive capacity of glioma cell lines was assessed and found to be similar to transplanted tumours previously reported in the literature. This suggests that if substantial numbers of viable hypoxic cells were present in situ in gliomas, then nitroimidazole-binding techniques should be capable of identifying them. Inter-tumour variability in the amount of necrosis was seen. M006 xenografts growing in subcutaneous and intracranial sites revealed extensive necrotic regions histologically, some of which were surrounded by cells labelled heavily for [3H]misonidazole, while other areas were lightly labelled. Similar binding patterns were seen for subcutaneous M059K tumours, while subcutaneous M01 Ob tumours display necroses of which almost all were surrounded by heavily labelled cells. The oxygen consumption rates of tumour cell lines grown in vivo, in which venous P02 concentrations are of the order of 2-5%, were two to sevenfold less than those of the same lines grown as monolayers in vitro under oxygen concentrations of 18%. We postulate that glioma cell lines behave as 'oxygen conformers', in that their rate of oxygen consumption appears to vary with the availability of oxygen. Together with the misonidazole-binding data, the results in this glioma tumour model are consistent with coordinate inhibition or down-regulation of respiration under moderate hypoxia.

“…Garrecht and Chapman (1983) noted the presence of significant '4C-misonidazole binding to murine liver, nasal and oral mucosa, as well as to implanted EMT6 tumours growing in BALB/c mice. Detailed studies using both scintillation counting and autoradiography have shown significant retention of misonidazole in many different murine tissues, including oesophagus, airway epithelium, liver, foot pad, eyelid (meibomian gland), sebaceous glands, stomach, and parotid gland (Akel et al, 1986, Cobb et al, 1989, 1990aMacManus et al, 1989). It could be argued that at least one of these tissues, liver, contains hypoxic cells (Van OsCorby & Chapman, 1986Maxwell et al, 1989;MacManus et al, 1989).…”

mentioning

confidence: 99%

Nitroimidazole adducts as markers for tissue hypoxia: mechanistic studies in aerobic normal tissues and tumour cells

Parliament¹,

Wiebe²,

Franko³

1992

Summary Two aspects of the aerobic metabolism of nitroimidazole markers for hypoxia were investigated. Several normal murine tissues which are likely to be well oxygenated bind misonidazole at rates comparable to those of hypoxic regions in tumours. The possibility that this aerobic activation occurs via an oxygen independent process such as an initial two electron reduction was studied. Binding to the oesophageal mucosa of mice which occurred under hypoxia in vitro was inhibited by at least 95% in the presence of 10% oxygen. Dicoumarol, an inhibitor of DT-diaphorase, was shown to cause only small reductions in misonidazole binding to oesophageal epithelium and smooth muscle in vitro and to EMT6 tumours, liver, oesophageal and tracheal epithelium, parotid gland and smooth muscle in vivo. Thus an oxygen-insensitive process is not a major cause of the high binding rate in oesophageal mucosa, and may not contribute significantly to the observed binding in other normal tissues. It has been suggested that metabolism of nitroimidazoles by aerobic cells in tumours might be sufficient to minimise access of these compounds to hypoxic regions, particularly at the micromolar concentrations currently in use clinically. The uptake of '251-iodoazomycin arabinoside by RIF-1 and EMT6 tumours was found to be directly proportional to injected dose over concentrations between 0.5 and 501M. Labelling of hypoxic regions in EMT6 tumours by high specific activity 3H-misonidazole at