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Biomedical subjects

J A Raleigh

Publications and source records attributed to J A Raleigh.

At least 19 recordsLinked to original sources
PubMed1992

Development of an ELISA for the detection of 2-nitroimidazole hypoxia markers bound to tumor tissue.

Canine and rodent tumors covalently bind the fluorinated 2-nitroimidazole, CCI-103F, in a way that immunohistochemical analysis shows is consistent with the location of tumor hypoxia. We have now developed a rapid, quantitative, and non-radioactive enzyme linked immunosorbent assay for the binding of CCI-103F in biopsy samples of spontaneous canine tumors. Issues of antigen stability during tissue processing, calibration of the ELISA, and the use of biopsy samples for measuring tumor hypoxia by the ELISA approach are addressed.

Animals↗
PubMed1992

Evaluation of a fluorinated 2-nitroimidazole binding to hypoxic cells in tumor-bearing rats by 19F magnetic resonance spectroscopy and immunohistochemistry.

We have examined a hexafluorinated 2-nitroimidazole, CCI-103F, as a probe for hypoxic tumor cells by in vivo 19F magnetic resonance spectroscopy (MRS). Following initial intraperitoneal injections of the drug in tumor-bearing (Dunning R3327-AT1-Matlylu) rats, 19F spectra were obtained on an Otsuka 2.0T Vivospec spectrometer using a 1.5-cm surface coil. Signal at 1- and 2-h time points indicated initial biodistribution of drug in the tumor. At 4 and 8 h, a progressive increase in signal intensity was observed, indicating retention of drug within the tumor. Tumor signal remained detectable in 4 of 10 rats at 24 h, indicating possible nitroreductive bioactivation by hypoxic cells. Immunohistochemistry of these tumors revealed a staining pattern consistent with labeling of hypoxic cells. No detectable 19F signal was found at 24 h for the other rats, indicating complete washout of unbound drug. Immunohistochemical assessment of these tumors revealed some staining for bound drug at the periphery of necrotic zones. 31P-MRS of the tumors showed good correlation with the presence or absence of hypoxia as evaluated by 19F-MRS, T1- and T2-weighted images, and immunohistochemistry. These results provide the groundwork for further studies using this misonidazole analog for noninvasive identification of hypoxic tumor cells in vivo by MRS.

Adenocarcinoma↗
PubMed1991

Radiolytic reduction of protein and nonprotein disulfides in the presence of formate: a chain reaction.

We have reported recently that the disulfide groups in bovine serum albumin can be reduced by a radiolytic chain reaction which occurs in deoxygenated solutions containing formate ions. This reaction, which involves the reduction of disulfide groups by hydrated electrons and carbon dioxide radical anions, has now been studied in greater detail and compared with an analogous reaction in small, disulfide containing molecules over a range of pH values and substrate concentrations. A two-step reaction is proposed to account for the reduction of disulfides in reactions which can have chain lengths of 20 or more. Thiols produced by the disulfide reduction are stable to the conditions of the reaction. For example, a biological assay showed that the integrity of glutathione was maintained even at radiation doses much larger than those required to achieve complete reduction of glutathione disulfide. It was found that the extent of disulfide reduction could easily be controlled by varying the radiation dose delivered to the solutions. Radiolytic reduction is a very useful way of reducing protein and low molecular weight disulfides without the use of excess quantities of reagents such as dithiothreitol. In many cases, the reaction solutions could be used directly for subsequent reactions and this may be of considerable value in modifying the structure of hormones, enzymes, membrane receptors, and other disulfide containing proteins. If ammonium formate is used, freeze drying is an effective way to remove the formate salt, should this be required.

Cesium Radioisotopes↗
PubMed1991

Development of an in vivo 19F magnetic resonance method for measuring oxygen deficiency in tumors.

A 19F magnetic resonance spectroscopy (MRS) approach to measuring hypoxia in experimental tumors in rats at a field strength of 2.35 T has been investigated in a combined study of in situ and excised tumors. The detection of tumor hypoxia is based on the hypoxia marker approach which depends on the selective, covalent binding of a fluorinated 2-nitroimidazole to hypoxic cells. The 19F MRS measurement of in vivo hypoxia marker binding was made at a fixed postinjection time when unbound, circulating marker molecule concentrations had dropped below detectable levels. A correlation between 19F MRS and scintillation counting measures of tumor-bound, tritium-labeled hypoxia marker was observed. There was no correlation between integrated 19F MRS hypoxia marker signals and the in vivo 31P MRS parameters of hypoxia which have been developed to measure normal tissue ischemia. Radiolabeling studies and previous immunohistochemical studies with the fluorinated hypoxia marker support the conclusion that the 19F MRS approach has promise as a physically noninvasive guide to the use of hypoxia-dependent therapies at clinically usable MRS field strengths.

Adenocarcinoma↗
PubMed1990

Importance of thiols in the reductive binding of 2-nitroimidazoles to macromolecules.

Reductive activation of 2-nitroimidazoles in the presence of bovine serum albumin (BSA) led to binding of the nitroheterocycles to the protein. The binding was most efficient to BSA in which protein disulfides had been reduced to thiol groups. Protein thiols were at least twenty times more efficient than other protein, RNA or DNA nucleophiles in binding the reductively-activated nitroheterocycles. This result is of practical importance in the development of immunoassays for 2-nitroimidazoles as hypoxia markers in normal and tumor tissue.

Glyoxal↗
PubMed1990

Immunohistochemical detection of a hypoxia marker in spontaneous canine tumours.

An immunoperoxidase technique has been used to detect the in vivo binding of a 2-nitroimidazole hypoxia marker in histochemical sections of a variety of excised canine tumours. The binding occurred 10-12 cell diameters away from tumour blood vessels, consistent with the expected location of hypoxic cells in tissues in which oxygen concentration gradients are established by diffusion. Hypoxic fractions ranging from 4 to 13% have been estimated on the basis of morphometric analysis of multiple tumour sections. The binding of the marker was restricted to the cytoplasm of the cells. The marker appeared in regions adjacent to necrosis but also in regions free of necrosis. As in earlier autoradiography studies, binding was occasionally observed in cells adjacent to tumour blood vessels. Generally, binding to normal tissues was not observed. However, binding to smooth muscle cells surrounding arterioles in some sections of normal tissue and tumour tissue was observed.

Animals↗
PubMed1990

Dynamic measurements of hexafluoromisonidazole (CCI-103F) retention in mouse tumours by 1H/19F magnetic resonance spectroscopy.

Selective retention of hexafluoromisonidazole, CCI-103F, in RIF-1 and SCCVII tumours of C3H/Km mice has been measured by 1H/19F magnetic resonance spectroscopy (MRS) on a Bruker AM-400 multinuclear spectrometer. CCI-103F concentrations in tumours and in normal tissues were measured using an MRS technique in which the water component in the tissues serves as an internal concentration reference. The biodistribution and elimination half-life of the drug in the tissues after i.p. injections were determined. The plasma half-life of the drug (41 min) was measured by high-pressure liquid chromatography. The two tumour lines and liver have longer retention times with half-lives of 47, 129 and 81 min, respectively, while normal tissues, muscle and brain have little retention of CCI-103F and clear the drug very quickly. Dynamic measurements of CCI-103F retention in tumours by MRS may provide a non-invasive probe for assessing tumour hypoxia.

Animals↗
PubMed1989

Metabolic binding of misonidazole to mouse tissues. Comparison between labels on the ring and side chain, and the production of tritiated water.

The 2-nitroimidazole, misonidazole, is of current interest as an imaging agent for hypoxic regions in tumors and in vascular disease such as stroke. The basis of this technique is the reductive activation and binding of nitroheterocycles which is much more efficient in the absence of oxygen. The appropriate molecular location for an active isotope on the nitroheterocyclic probe depends on the nature of the metabolites retained in tissues after the parent drug has been cleared. Previous studies with tumor cells in vitro indicated that a ring label (2-14C) and a side-chain label (3H) were retained equally efficiently in the acid-insoluble fraction, whereas 1.5 to 3 times more side-chain label was retained in the total pool (acid soluble plus acid insoluble) of metabolites in several normal murine tissues. We show here that the excess side-chain label in six normal tissues, plasma and EMT6 tumors was found entirely in the acid-soluble fraction as a volatile component. This volatile component was tentatively identified as tritiated water. It appeared that, in general, molecular products of misonidazole metabolism were retained in mouse tissues, with the exceptions that a small excess of ring label was found in liver and heart and that tritiated water appeared in the acid-soluble fraction of all tissues. Tritiated water would not be important in imaging studies but could be a factor in studies in which scintillation counting of tritiated nitroheterocyles is used.

Aerobiosis↗
PubMed1989

Quantitation of hypoxia in multicellular spheroids by video image analysis.

Polyclonal antibodies to a series of radiation chemically produced 2-nitroimidazole-protein adducts have been used in the indirect immunofluorescent detection of hypoxic cells in EMT6/Ed spheroids. The spheroids were incubated with the radioactive 2-nitrioimidazoles under atmospheres of air, several intermediate oxygen concentrations, and nitrogen. The fluorescence intensity of the marker across radii of spheroids labeled at various oxygen tensions, as measured by video image analysis, paralleled the autoradiographic grain density. Thus, we conclude that the immunohistochemical approach provides a quantitative measure of the distribution of 2-nitroimidazole adducts in spheroids. The advantages of speed, technical simplicity, economy, and independence from the requirement of radiolabeled precursor render the fluorescent marker of potential use in the clinical detection of cellular hypoxia and tissue ischemia.

Autoradiography↗
PubMed1988

Oxygen dependence of product formation in irradiated adenosine 5'-monophosphate.

The formation of (R)- and (S)-8,5'-cycloadenosine 5'-monophosphate (8,5'-cycloAMP), 8-hydroxyadenosine 5'-monophosphate (8-hydroxyAMP), and radiolytic adenine release from irradiated solutions of adenosine 5'-monophosphate (5'-AMP) was measured as a function of increasing liquid-phase oxygen concentration. Three classes of specific molecular damage were identified on the basis of the oxygen dependence for product formation. Major changes in product yield occurred near the range of oxygen concentrations associated with the radiobiological oxygen effect. In addition to these data, systematic increases in the concentration of hydrogen peroxide at the time of irradiation resulted in an increase in the yield of 8-hydroxyAMP and a component of radiolytic adenine release in nitrogen-saturated solutions of 5'-AMP. However, no changes in the yield of the 8,5'-cyclonucleotides were observed under these conditions.

Adenine↗
PubMed1987

Fluorescence immunohistochemical detection of hypoxic cells in spheroids and tumours.

Polyclonal antibodies have been raised in rabbits to a haemocyanin adduct of a reductively-activated, fluorinated analogue of misonidazole. Fluorescence immunohistochemical studies show that the polyclonal antibodies bind to spheroid sections and tumour sections in patterns similar to those revealed by autoradiographic studies with a tritium-labelled derivative of the fluorinated misonidazole analogue.

Animals↗
PubMed1987

Interaction of nitroaromatic radiosensitizers with irradiated polyadenylic acid as measured by an indirect immunochemical assay with specificity for the 8,5'-cycloadenosine moiety.

The relative reactivity of a series of nitroaromatic radiosensitizers toward the C(5') radical intermediate leading to 8,5'-cycloadenosine formation in deoxygenated solutions of irradiated polyadenylic acid (poly A) was assessed using standard competition kinetic analysis. Formation of 8,5'-cycloadenosine was assayed by an indirect, competitive, enzyme-linked immunosorbent assay (ELISA) described in an earlier report. In the absence of oxygen, the nitroaromatics inhibit 8,5'-cyclonucleoside formation in a way which generally increases with radiosensitizer electron affinity. Although hydroxyl radical scavenging by the nitroaromatics may account for a relatively small decrease in 8,5'-cyclonucleoside formation, the data suggest that oxidation of the C(5') radical intermediate is the more plausible explanation for the decreased yield of the 8,5'-cyclonucleoside with increasing nitroaromatic electron affinity.

Adenosine↗
PubMed1987

Intramolecular cyclization in irradiated nucleic acids: correlation between high-performance liquid chromatography and an immunochemical assay for 8,5'-cycloadenosine in irradiated poly(A).

A correlation between high-performance liquid chromatography (HPLC) analysis and an in situ enzyme-linked immunosorbent assay (ELISA) for 8,5'-cycloadenosine formation in irradiated poly(A) has been established. The correlation shows that the ELISA precisely reflects changes in the combined yield of R- and S-8,5'-cycloadenosine but that a correction factor must be applied to the ELISA values for accuracy. The HPLC analysis reveals that the intramolecular cyclization proceeds stereoselectively in irradiated poly(A) to preferentially produce the R isomer at pH 7.0 which is similar to the result for irradiated adenosine but in contrast to the result for 5'-AMP where the S isomer predominates at neutral pH. The HPLC analysis shows that two events originating in hydroxyl radical attack at the sugar phosphate backbone in poly(A); that is, adenine release and 8,5'-cycloadenosine formation have somewhat different dose-yield responses. The formation of 8-hydroxyadenosine was detected in the HPLC chromatograms of poly(A) irradiated under N2O at neutral pH, and the yield of this compound was similar to the yield observed in 5'-AMP or adenosine irradiated under similar conditions.

Adenosine↗
PubMed1986

Stereoselective intramolecular cyclization in irradiated nucleic acids: R- and S-8,5'-cycloadenosine in polyadenylic acid.

The yields of R- and S-8,5'-cycloadenosines have been measured in poly A irradiated with gamma rays in the absence of oxygen. High performance liquid chromatographic analysis of the nucleoside analogues obtained by hydrolysis of the irradiated poly A shows that the R isomer predominates to the extent of 2.5-fold at doses of ionizing radiation in the range of 0-400 Gy.

Adenosine↗
PubMed1986

Covalent binding of a fluorinated 2-nitroimidazole to EMT-6 tumors in Balb/C mice: detection by F-19 nuclear magnetic resonance at 2.35 T.

A hexafluorinated 2-nitroimidazole (CCI-103F) has been synthesized and its properties as a hypoxic cell binder investigated. The drug has a plasma half-life of 90 min in Balb/C mice and a tumor-to-plasma ratio of 0.8. Following sustained exposure to the drug and a washout period for the unbound drug, liver and tumor samples were excised. NMR investigation of the excised tissue in a 2.35 T animal sized NMR facility revealed the presence of 19F bound to both tissues. Further improvement in sensitivity is required to make 19F NMR detection of binding in situ feasible.

Animals↗