Relationship between electrochemical gradients and cellular permeability to misonidazole

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<ul><li><p>O3CU-3Dlh/BJ 103 00 + 00 C op)nghl cc IO84 Pcrgamon Press Ltd </p><p>??Session II Abstracts </p><p>RELATIONSHIP BETWEEN ELECTROCHEMICAL GRADIENTS AND CELLULAR PERMEABILITY </p><p>TO MISONIDAZOLE </p><p>R. GONZALEZ-MENDEZ, D. M. BROWN, J. M. SAWYER, G. M. HAHN AND J. M. BROWN </p><p>Division of Radiobiology Research, Department of Radiology Stanford University School of Medicine, Stanford, CA 94305 </p><p>Alterations in pH and temperature or agents such as the protonophore carbonyl cyanide-3-chlorophenyl hydrazone (CCCP) modify the cellular proton electrochemical gradient or proton motive force (P+). The proton electrochemical gradient is defined as: </p><p>where: F = Faradays constant; A* = membrane potential; R = gas constant; and T = K. </p><p>The purpose of this study was 10 determine whether a relation occurs between the enhanced intracellular uptake of misonidazole observed under hyperthermia, acidic pH or in the presence of CCCP (Brown, D.M., el al.: Radial. Res. 93: 492, 1983) and changes in pH+. </p><p>The membrane potential difference (AO) was measured by inducing a 86Rb diffusion potential with the ionophore valinomycin while the intracellular pH was measured using P-nuclear magnetic resonsance (NMR) and weak acid partitioning in 5,5 dimethyl oxazolidine 2,4 dione (DMO). </p><p>The A* measured in suspensions of aerobic HA- I cells at 37C was -60 mV. With the cells at 37C. variations in the extracellular pH from 6.0 to 7.7 did not significantly alter A*. However, at temperatures of 43C for up to I hr., the A* rose to -75 mV while in the presence of CCCP, 0 mV was observed. </p><p>When AF+ was zero (i.e., in the presence of CCCP) 100% intracellular uptake of MIS0 was observed. Surprisingly, when AF. was increased, as the external pH was lowered, MIS0 intracellular uptake also increased suggesting that alterations in Ati. will enhance MIS0 uptake. However, at elevated temperatures, Ac(,,. remains relatively constant at - -50 mV even though MIS0 intracellular uptake increases. This may be partially explained by the alterations in the ionic environment inside the cells as indicated by a higher A* and lower pH, at elevated tem- peratures. These results imply that the potential differences that exist across the plasma membrane are barriers for drugs that enter the cell by passive diffusion. </p><p>(Supported by NIH Grants CA I5201 and CA 04542.) </p><p>THE INTERACIION OF TUMOR LOCALIZING PORPHYRINS WITH COLLAGEN, ELASTIN, FIBRIN, GELATIN, </p><p>AND FIBRINOGEN </p><p>M. A. EL-FAR AND N. R. PIMSTONE University of California, Davis, School of Medicine. Department of </p><p>Internal Medicine, Davis, CA 956 16, USA </p><p>We were the first to show that uroporphyrin I (Uro I) is a superior tumor local&amp; when compared lo haematoporphyrin derivative (HPD) and thereof photofrin II (SPP) [C/in. Rex (1983) 31: 21. This study addresses the ability of various porphyrins lo bind certain proteins which </p><p>may be found in tumor stroma. The tumor localizing porphyrins studied were HPD, SPP, Uro I and Uro III (uroporphyrin isomer III). Copro- porphyrin III (CP III) and deuteroporphyrin IX (DP) were included as non-tumor localizing porphyrins. The interaction of this series of por- phyrins with acid soluble collagen (ASC) and acid insoluble (AIC). elastin (E), and fibrin (F) was determined. Briefly, 5 mg quantities of E, ASC. AIC and F were incubated with 3.0 ml of various porphyrins at different concentrations and for varying periods in the dark at 37C. After in- cubation, the percent unbound porphyrins of each was determined flu- orometrically. Also, the binding affinity of Uro I, Uro III and DP 1X to gelatin with different bloom numbers (60. 175, and 300) and fibrinogen was determined spectrofluorometrically using an Amicon CF-25 mem- brane cone as previously used for albumin binding by El-Far and Pim- stone [Gel/ Biochemistry and Funnion (1983): 21. Uro 1 and III showed highest binding affinity to elastin while HPD and SPP showed not only the lowest binding affinity but also the same affinity shown by non- tumor localizing porphyrins as CP III and DP IX. Only the tumor localizing porphyrins showed significant binding lo collagen especially with acid soluble type. Indeed, Uro I showed the highest binding. A similar finding was noted with gelatin (which is known as denatured collagen) especially with lower bloom numbers. HPD and SPP showed a more significant binding 16 fibrin than that shown by Uro I and III. while non-tumor localizing porphyrins did not. The results suggest that collagen and fibrin may play a significant role in prophyrin localization in tumor cells. The fact that malignant epithelial cells oftumon produce collagen and contain the key enzyme in collagen biosynthesis would provide a notion that collagen is possibly one of the key factors which govern the tumor uptake of prophyrins. While elastin and fibrinogen by themselves do not appear to participate. Understanding the specific porphyrin interactions with the stroma constituents will help in the search for an ideal localizer and sensitizer. </p><p>LACTIC ACID IN TUMOURS: AN INDICATOR OF HYPOXIC FOCI? </p><p>J. S. MAHOOD AND R. 1. WILLSON Dept. Biochemistry, Brunei University. </p><p>Uxbridge, Middlesex. UB8 3PH </p><p>We have previously shown that L.(+) lactic acid can enhance the cytotoxic action of the nitroimidazole radiosensitizen metronidazole and mison- idazole (Mahood, J.S., Willson, R.L.: Cytotoxity of metronidazole (flagyl) and misonidazole (RO-07-0582): Enhancement by lactate. Br. J. Cancer 43, 350). We have now investigated the presence of L(+) lactic acid in solid turnours and have developed a staining method which may identify hypoxic foci. </p><p>Complete solid turnours are sectioned on a cryomicrotome after tlash freezing in liquid nitrogen. Following freeze drying sections are stained for lactic acid by immening in a mixture of tetrazolium violet and yeast lactate dehydrogenase (cytochrome b2) at pH 8.4. After drying sections are counter stained in malachite green to improve the optical resolution. Stained sections are observed on a Zeiss photomicroscope fitted with an Optomax T.V. camera linked lo an image analyser and a computer. </p><p>In a preliminary study of mouSe turnours of radiobiological interest results indicate that in the W203033 adenocarcinoma up to 32% of the tumour volume contains relatively high concentrations of lactic acid. In the case of the squamous carcinoma Da value of approximately 20% was obtained which compares lo a value of 19% for the radiobiological fraction for this tumour. </p><p>I799 </p></li></ul>

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