Intracellular pH recovery from lactic acidosis of single skeletal muscle fibers

Intracellular pH (pHi), measured with H+-selective microelectrodes, in quiescent frog sartorius muscle fibres was 7.29 +/- 0.09 (n = 13). Frog muscle fibres were superfused with a modified Ringer solution containing 30 mM HEPES buffer, at extracellular pH (pHo) 7.35. Intracellular pH decreased to 6.45 +/- 0.14 (n = 13) following replacement of 30 mM NaCl with sodium lactate (30 mM MES, pHo 6.20). Intracellular pH recovery, upon removal of external lactic acid, depended on the buffer concentration of the modified Ringer solution. The measured values of the pHi recovery rates was 0.06 +/- 0.01 delta pHi/min (n = 5) in 3 mM HEPES and was 0.18 +/- 0.06 delta pHi/min (n = 13) in 30 mM HEPES, pHo 7.35. The Na+-H+ exchange inhibitor amiloride (2 mM) slightly reduced pHi recovery rate. The results indicate that the net proton efflux from lactic acidotic frog skeletal muscle is mainly by lactic acid efflux and is limited by the transmembrane pH gradient which, in turn, depends on the extracellular buffer capacity in the diffusion limited space around the muscle fibres.     

Radiosensitization of Chinese hamster cells by oxygen and misonidazole at low X-ray doses

The radiosensitization of Chinese hamster V79 cells in vitro by air and misonidazole at low X-ray doses (0.2-6.0 Gy) had been studied. These survival data, together with high-dose data, were fitted to the linear quadratic model ln S = -(alpha D + beta D2), deriving estimates of alpha and beta by six different methods to illustrate the influence of the statistical treatment on the values so derived. This in vitro study clearly demonstrated that the survival parameters alpha and beta are dependent to some degree on the method of analysis of the raw survival data; however, their ratios, the values of oxygen enhancement ratios (OERs) and radiosensitizer enhancement ratios (SERs) derived from the different methods, are similar. All methods of analysis give reduced OERs at low radiation doses for combined low- and high-dose X-ray data. However, the OERs are still appreciably high, ranging from 2.45 to 2.50 for an oxic dose of 2 Gy. All methods of analysis gave reduced SERs at low doses for combined low and high X-ray dose data for hypoxic cells irradiated in 1 mmol dm-3 misonidazole. At survival levels corresponding to doses of 2 Gy in the presence of 1 mmol dm-3 misonidazole and SERs ranged from 1.2 to 1.5.     

Regulation of intracellular pH in human neutrophils

The intracellular pH (pHi) of isolated human peripheral blood neutrophils was measured from the fluorescence of 6-carboxyfluorescein (6-CF) and from the equilibrium distribution of [14C]5,5-dimethyloxazolidine -2,4-dione (DMO). At an extracellular pH (pHo) of 7.40 in nominally CO2-free medium, the steady state pHi using either indicator was approximately 7.25. When pHo was suddenly raised from 7.40 to 8.40 in the nominal absence of CO2, pHi slowly rose by approximately 0.35 during the subsequent hour. A change of similar magnitude in the opposite direction occurred when pHo was reduced to 6.40. Both changes were reversible. Intrinsic intracellular buffering power, determined by using graded pulses of CO2 or NH4Cl, was approximately 50 mM/pH over the pHi range of 6.8-7.9. The course of pHi obtained from the distribution of DMO was followed during and after imposition of intracellular acid and alkaline loads. Intracellular acidification was brought about either by exposing cells to 18% CO2 or by prepulsing with 30 mM NH4Cl, while pHo was maintained at 7.40. In both instances, pHi (6.80 and 6.45, respectively) recovered toward the control value at rates of 0.029 and 0.134 pH/min. These rates were reduced by approximately 90% either by 1 mM amiloride or by replacement of extracellular Na with N-methyl-D-glucamine. Recovery was not affected by 1 mM SITS or by 40 mM alpha-cyano-4-hydroxycinnamate (CHC), which inhibits anion exchange in neutrophils. Therefore, recovery from acid loading is probably due to an exchange of internal H for external Na. Intracellular alkalinization was achieved by exposing the cells to 30 mM NH4Cl or by prepulsing with 18% CO2, both at a constant pHo 7.40. In both instances, pHi, which was 7.65 and 7.76, respectively, recovered to the control value. The recovery rates (0.033 and 0.077 pH/min, respectively) were reduced by 80-90% either by 40 mM CHC or by replacement of extracellular Cl with p-aminohippurate (PAH). SITS, amiloride, and ouabain (0.1 mM) were ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of bronchial arterial PO2 on pulmonary vascular resistance

In six anesthetized and mechanically ventilated adult sheep, the bronchial artery was perfused with blood from an oxygenator-pump circuit. When the lungs were ventilated with 100% O2 and the bronchial O2 tension (PbrO2) was approximately 600 Torr, the mean of the pulmonary vascular resistances (PVR) measured at the beginning (3.32 +/- 0.29 units) and end (3.17 +/- 0.13 units) of the experiment was 3.24 +/- 0.20 units. When the PbrO2 was changed to 58 +/- 1 Torr, the PVR (2.99 +/- 0.14 units) did not change significantly. However, when the lungs were ventilated with air as PbrO2 was decreased to 91 +/- 4, 77 +/- 3, 56 +/- 2, and 42 +/- 1 Torr, the PVR increased to 3.67 +/- 0.18, 4.03 +/- 0.16, 4.79 +/- 0.19, and 4.71 +/- 0.35 units, respectively. However, when the PbrO2 was decreased further to 26 +/- 1 and 13 +/- 1 Torr, the PVR decreased to 3.77 +/- 0.28 and 3.91 +/- 0.30 units, respectively. In contrast, the bronchial vascular resistance decreased monotonically as PbrO2 decreased. The bronchial circulation supplies vasa vasorum to the walls of all but the smallest pulmonary arteries, and it is therefore suggested that the PO2 of the bronchial circulation is responsible for the bimodal response of the pulmonary vasculature, with stimulation of hypoxic pulmonary vasoconstriction at moderate hypoxemia and of hypoxic pulmonary vasodilation at profound hypoxemia. The physiological and pathophysiological significance of the influence of systemic PO2 on pulmonary vascular tone is discussed.     

The effect of extracellular pH on radiosensitization by misonidazole and acidic or basic analogues

The effect of extracellular pH (pHe) on the radiosensitization of hypoxic Chinese hamster V79 cells in vitro by the 2-nitroimidazole, misonidazole, and analogues substituted with basic or acid functions has been studied. Misonidazole (1 mmol dm-3) gave an enhancement ratio (e.r.) of 1.6 which remained unchanged over the pHe range of 3.8-9.5. Control hypoxic survival curves in the absence of sensitizer also remained essentially unchanged over this pHe range. These results contrast with those seen for 0.1 mmol dm-3 Ro 03-8799 (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanol), a base with pKa = 8.9): the ER increased from 1.4 to 2.1 as pHe increased from 5.6 to 8.4. However, with the weaker bases, Ro 03-8800 and nimorazole (morpholino derivatives with pKa = 6.3 and 5.2 respectively) the e.r. remained constant over a wide pHe range. Nitroimidazoles substituted with acidic functions gave decreasing sensitization with increasing pHe. For azomycin (pKa = 7.2) at 1 mmol dm-3 the e.r. decreased from 1.9 at pHe 4 to 1.0 at pHe 9. The effect of the proton conductor carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 10 mumol dm-3) on radiosensitization by Ro 03-8799 (0.1 mmol dm-3) and misonidazole (1.0 mmol dm-3) was also studied. At pHe 6.67 the e.r. for Ro 03-8799 was increased from 1.36 to 1.76 by the presence of CCCP, whereas at pHe 7.33 the e.r. was unchanged. In contrast the e.r. for misonidazole was unchanged at pHe 6.65 and 7.33. These results are consistent with pH differentials across the cell membrane creating intracellular:extracellular concentrations gradients for radiosensitizers with acidic or basic functions.     

Radiosensitization, pharmacokinetics, and toxicity of a 2-nitroimidazole nucleoside (RA-263)

A 2-nitroimidazole nucleoside, 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimida zole (RA-263), has been investigated for its radiosensitization, pharmacokinetics, and toxicity properties. The in vitro radiosensitization tests against hypoxic Chinese hamster (V-79) cells demonstrated that RA-263 was a more potent radiosensitizer than misonidazole and at 2 mM concentration approached the oxic curve. Significant in vitro radiosensitization activity was also observed in EMT6 mammary tumor cells. The in vitro cytotoxicity data suggested that RA-263 is considerably more toxic to hypoxic cells than misonidazole. The increased cytotoxicity may be related to its higher depletion of nonprotein thiols (NPSH) than misonidazole. The combined effects of radiosensitization and hypoxic cell toxicity were measured by preincubation of the V-79 cells for 4 h under hypoxic conditions before irradiation. The results demonstrated a synergistic response by causing a significant decrease in the extrapolation number with loss of shoulder of the radiation survival curves. The in vivo radiosensitization experiments conducted by the in vivo-in vitro cloning assay with the EMT6 mammary tumor indicate that RA-263 is an effective sensitizer. Pharmacokinetic data suggested that RA-263 was eliminated from plasma by a rapid alpha phase and a slower beta phase with T 1/2 of 36 and 72 min, respectively. The concentration in the brain was approximately one-sixth of tumor concentration, suggesting that RA-263 is excluded from the CNS. Moreover, RA-263 was two times less toxic than misonidazole on equimolar basis by acute LD50 tests. This agent was also significantly less mutagenic than misonidazole in a strain of Escherichia coli.     

Interaction of platinum(II) tetrachlorodianion (Fast Black)2 with superhelical DNA and with radiation in vitro and in vivo

A new complex of tetrachloroplatinum(II) and the azoic diazo dye, Fast Black K, Pt(Fast Black)2, was made in an attempt to produce an uncharged molecule which could readily gain access into cells and could bring a high concentration of tetrachloroplatinum into the vicinity of the DNA. Even the lowest concentration of Pt(Fast Black)2 tested in the superhelical pBR322 plasmid DNA assay in vitro completely converted the superhelical DNA to the circular and linear forms by 24 h. When the cytotoxicity of the Pt(Fast Black)2 and Fast Black were tested in exponentially growing EMT6 cells. Pt(Fast Black)2 was slightly more toxic to normally oxygenated than to hypoxic cells at pH 7.40, but was far more toxic to cells at pH 6.45 with no difference based on cellular oxygenation. Fast Black was much less toxic than Pt(Fast Black)2 and its cytotoxicity was unaffected by pH. Pt(Fast Black)2 had a small radiosensitizing effect on hypoxic EMT6 cells with a dose-modifying factor of 1.3, but exposure to the drug entirely removed the shoulder region on the radiation survival curves for both the oxygenated and hypoxic cells. In contrast, Fast Black reduced the shoulder in hypoxic but not in oxygenated cells. When Pt(Fast Black)2 (500 mg/kg), Fast Black (300 mg/kg) (the maximally tolerated dose), or misonidazole (1 g/kg) were given intraperitoneally 15 min prior to irradiation of FSaIIC tumors with 0, 10, 20, or 30 Gy, Pt(Fast Black)2 alone caused a tumor growth delay of 6 days versus 3 days for Fast Black. With radiation, Pt(Fast Black)2 produced the greatest enhancement in tumor growth delay of the drugs tested, especially at the lowest (10 Gy) radiation dose (i.e., in the in vivo "shoulder region"). These results indicate that Pt(Fast Black)2 may be suitable for clinical development because it causes both significant direct cytotoxicity and enhancement of radiation killing. The fact that its cytotoxicity is markedly increased at an acidic pH and its radiation enhancing effects are greatest in combination with relatively low single-fraction radiation doses make it especially interesting. The cytotoxicity of Pt(Fast Black)2 may be influenced by the tumor environment, and the radiosensitizing properties appear well suited for use with radiation fraction sizes that are employed in the clinic.     

Radiosensitization of hypoxic HeLa S3 cells in vitro by a new type of radiosensitizer: spermine and spermidine amides with nitro groups

A new type of hypoxic cell sensitizer (FNT-series compounds) has been developed and tested on HeLa S3 cells. They have two nitrobenzoyl groups at both ends of the spermine or spermidine in their chemical structures. Their ability to sensitize hypoxic cells is greater than that of misonidazole. Among them, N1, N10-bis(4-nitrobenzoyl)-spermidine (FNT-1) was most effective. 1 mM FNT-1 gave a corrected enhancement ratio of 1.71 compared to 1.32 for the same concentration of misonidazole. Its electron affinity, in terms of the half-wave reduction potential, was - 350 mV and higher than that of misonidazole (-395 mV). These FNT-series compounds are thought to interact with DNA in two ways; noncovalent linkage between the basic groups of the polyamine and highly acidic phosphate moieties of the nucleic acid and, secondly, the insertion of the nitrobenzoyl groups between base pairs of the DNA double helix. Since spermine and spermidine themselves did not show any sensitizing activities, it is suggested that the nitrobenzoyl groups which are introduced in spermine and spermidine, play an important role in sensitization.     

Phosphorus-31 nuclear-magnetic-resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver

A quantitative analysis of the phosphorus-31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30-mm sample cell. The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of Pi and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well-oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for Pi and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pHi) is 7.25 +/- 0.05 and free magnesium concentration 0.5 mM. Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and Pi levels, respectively. A large amount of sn-glycerol 3-phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic Pi is observed, which is indicative of a net uptake of Pi across the plasma membrane that occurred during ethanol oxidation. No significant pH variation is observed during ethanol infusion. In perfused liver of rats subjected to 48-h fasts, the concentrations of cytosolic phosphorylated metabolites are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and Pi, respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the Pi content is decreased by 10%. During a 4-min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min-1 (100 g body weight)-1, ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic Pi and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02 +/- 0.05 at the end of the 4-min hypoxic period.     

Priming effect of hyperosmotic stress on TRH-induced activation of Na+/H+ exchange in GH4C1 rat pituitary cells

The effect of mild hyperosmotic stress on cytosolic pH (pHi) alone, and in combination with thyrotropin-releasing hormone (TRH) or the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was investigated in GH4C1 cells at resting pHi. Hyperosmotic stress induced by addition of 50 mM choline was without an effect on pHi. In cells stimulated with either TRH or TPA after choline, pHi increased 0.15 +/- 0.05 and 0.14 +/- 0.03 pH units, respectively (mean +/- SD). A similar response was obtained if TRH or TPA was added prior to choline. The effect was abolished by replacing extracellular Na+ with choline+, and by pretreatment of the cells with amiloride, indicating that the change in pHi probably was dependent on activation of Na+/H+ exchange. The results thus indicate that, in GH4C1 cells, hyperosmotic stress in combination with TRH or TPA can activate Na+/H+ exchange at resting pHi levels.     

Characterization of amyloplastic phosphohexose isomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphohexose isomerase from amyloplasts of immature wheat endosperm was purified 133-fold. The enzyme had a molecular weight of 130 kDa and maximum activity at pH 8.6. It showed normal hyperbolic kinetics for both fructose-6-P and glucose-6-P with Km of 0.12 mM and 0.44 mM, respectively. pH had a great influence on Km for fructose-6-P. Using glucose-6-P as the substrate, the equilibrium was reached at 23% fructose-6-P and 77% glucose-6-P and an equilibrium constant of about 3.0. The delta F calculated from the apparent equilibrium constant was +742 cal.mol-1. The activation energy calculated from the Arrhenius plot was 7450 cal.mol-1. None of the sulphydryl reagents at 2.5 mM concentration inactivated the enzyme. The enzyme was competitively inhibited by 6-phosphogluconate, ribose-5-P and ribulose-5-P with Ki values of 0.18, 0.14, and 0.13 mM, respectively. The probable role of the enzyme in starch biosynthesis in amyloplasts is discussed.     

Sex differences in gastric mucosal protection after 16, 16-dimethyl PGE2 and lithium chloride

While the incidence of duodenal ulcer disease has been documented to be greater in men than in women, this observation has not been previously noted in animal studies of the upper gastrointestinal tract. In this study, we questioned whether the cytoprotective properties of 16, 16-dimethyl PGE2 were sex-related by comparing the degree of ethanol-induced hemorrhagic gastritis in male and female rats pretreated with 16,16-dimethyl PGE2 or lithium chloride. Animals receiving 16,16-dimethyl PGE2 or lithium chloride had significantly less ethanol-induced hemorrhagic gastritis (1.17 +/- 0.15 and 1.24 +/- 0.13, respectively, p less than 0.001) when compared with controls (2.69 +/- 0.10). Female rats treated with 16,16-dimethyl PGE2 had 59% less hemorrhagic gastritis than male rats treated similarly (0.76 +/- 0.14 vs 1.86 +/- 0.19 respectively, p less than 0.001). This sex-related difference in hemorrhagic gastritis was not noted in male and female rats receiving lithium chloride (1.24 +/- 0.15 vs 1.23 +/- 0.27, respectively). However, female rats treated with 16, 16-dimethyl PGE2 had significantly less hemorrhagic gastritis when compared with female rats receiving lithium chloride (0.76 +/- 0.14 vs 1.24 +/- 0.15 respectively, p less than 0.05).     

Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichomonas vaginalis: identification of a phospholipase A-dependent hemolytic activity in a vesicular subcellular fraction

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.     

A 31P-NMR study on the recovery of intracellular pH in LLC-PK1/Cl4 cells from intracellular alkalinization

The regulation of intracellular pH (pHi) in a renal epithelial cell line, LLC-PK1/Cl4, during re-acidification from an alkaline load was studied by 31P-NMR. Intracellular alkalinization was induced by 10 mM ammonium glucuronate or by preloading with and subsequent removal of 20% CO2; the rate of re-acidification was found to be 0.047 pH units/min and 0.053 pH units/min, respectively. This rate of re-acidification was inhibited by 83% if Cl- was removed from the extracellular medium. A similar inhibition was found in the presence of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (76% inhibition) and 1 mM bumetanide (81% inhibition). No change in recovery was found after removing sodium from the extracellular medium, indicating that LLC-PK1/Cl4 cells recover from an intracellular alkaline load by a Cl-/HCO3- exchanger, which is SITS- and bumetanide-sensitive and has no requirement for sodium. In addition, the steady-state pHi in Cl4 cells was monitored by 31P-NMR. Removal of Cl- from the extracellular medium introduced an increase in pHi by 0.33 pH units, whereas 1 mM SITS and 1 mM bumetanide caused an increase in pHi by 0.14 or 0.13 pH units. In the presence of 1 mM amiloride, an inhibitor of the Na+/H+ exchanger, the steady-state pHi did not change significantly. These results indicate that at pHo 7.4 the steady-state intracellular pH of LLC-PK1/Cl4 cells strongly depends on the activity of the Cl-/HCO3- exchanger. Under the same conditions the activity of the Na+/H+ exchanger seems to be negligible.     

Targeting radiosensitizers to DNA by attachment of an intercalating group: nitroimidazole-linked phenanthridines

The nitroimidazole-linked phenanthridine series of compounds (NLP-1, 2, and 3) were synthesized under the assumption that it should be possible to enhance the molar efficiency of 2-nitroimidazoles as hypoxic cell radiosensitizers and cytotoxins by targeting them to their likely site of action, DNA. The targeting group chosen was the phenanthridine moiety, the major component of the classical DNA intercalating compound, ethidium bromide. The sole difference between the compounds is the length of the hydrocarbon chain linking the nitroimidazole to the phenanthridine. The phenanthridine group with a three-carbon side chain, P-1, was also synthesized to allow studies on the effect of the targeting group by itself. The ability of the compounds to bind to DNA is inversely proportional to their linker chain length with binding constant values ranging from approximately 1 x 10(5) mol-1 for NLP-2 to 6 x 10(5) mol-1 for NLP-3. The NLP compounds show selective toxicity to hypoxic cells at 37 degrees C at external drug concentrations 10-40 times lower than would be required for untargeted 2-nitroimidazoles such as misonidazole in vitro. Toxicity to both hypoxic and aerobic cells is dependent on the linker chain: the shorter the chain, the greater the toxicity. In addition, the NLP compounds radiosensitize hypoxic cells at external drug concentrations as low as 0.05 mM with almost the full oxygen effect being observed at a concentration of 0.5 mM. These concentrations are 10-100 times lower than would be required for similar radiosensitization using misonidazole. Radiosensitizing ability is independent of linker chain length. The present compounds represent prototypes for further studies of the efficacy and mechanism of action of 2-nitroimidazoles targeted to DNA by linkage to an intercalating group.     

Cytotoxicity, radiosensitization, and DNA interaction of platinum complexes of thiazin and xanthene dyes

Complexes of the platinum(II) tetrachlorodianion with positively charged nuclear dyes have been prepared in an effort to produce neutral molecules which could gain ready access to the nuclear DNA where the platinum(II) tetrachlorodianion could function as a radiosensitizing and a bifunctional alkylating agent. The thiazin dyes Thionin, Azure B, and Methylene Blue, the aminoxanthene dye Pyronin Y, and the thiazole dye Thioflavin have each been complexed to the platinum(II) tetrachlorodianion(PtCl4) in a ratio of 2:1(dye:PtCl4). Studies of the interaction of these complexes and of the dyes with the pBR322 plasmid superhelical DNA demonstrated that while each complex and dye readily associated with the DNA in a dose-dependent manner, only Pt(Thioflavin)2 and Thioflavin produced irreversible DNA changes (single-strand breaks). In exponentially growing EMT6 cells the cytotoxicity of these drugs was assessed in normally oxygenated and hypoxic cells at both pH 7.4 and 6.45. At concentrations ranging from 1 to 500 microM, Pt(Methylene Blue)2 was significantly more cytotoxic than the other thiazin dye complexes Pt(Thionin)2 and Pt(Azure B)2. The cytotoxicity of Pt(Thionin)2 and Pt(Methylene Blue)2 was increased in normally oxygenated and hypoxic cells at low pH. Both Pt(Pyronin Y)2 and Pt(Thioflavin)2 were more toxic than the thiazin complexes. Pt(Pyronin Y)2 was most cytotoxic to normally oxygenated cells at normal pH and hypoxic cells at low pH, while Pt(Thioflavin)2 was most cytotoxic to cells at low pH under both oxygenation conditions. In vitro studies of the radiosensitizing properties of these agents in EMT6 cells demonstrated that exposure to 100 microM for 1 h before and during irradiation (except for Pt[Thioflavin]2, which was assayed at 25 microM) resulted in enhancement rations of 2.5, 1.9, 1.5, and 1.5 for Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y)2, and Pt(Thioflavin)2, respectively, in hypoxic cells. In contrast, Pt(Methylene Blue)2 (and Methylene Blue) proved to be a radioprotector of normally oxygenated cells and did not sensitize hypoxic cells to the cytotoxic effects of radiation. In the FSaIIC fibrosarcoma in vivo administration of each drug at 100 mg/kg intraperitoneally (ip) 15 min prior to irradiation (except for Pt[Thioflavin]2, which was given at 1 mg/kg ip) showed that, with single radiation fractions of 10 and 20 Gy, dose-modifying factors of 2.1, 1.8, 1.5, and 1.2 were produced by Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y), and Pt(Methylene Blue)2, respectively, after correcting for growth delays induced by the drug alone. In comparison, misonidazole at 1 g/kg ip produced a dose-modifying factor of 1.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of misonidazole on formation of thymine damage by gamma rays

The effect of the radiosensitizer misonidazole (Ro-07-0582) on the formation of thymine base damage of the 5,6-dihydroxydihydrothymine-type by gamma rays was measured under aerobic and hypoxic conditions. HeLa cells, prelabeled with [methyl-3H]thymidine, were suspended in phosphate-buffered saline in the presence and absence of misonidazole. Concentrations of misonidazole up to 15 mM were used. The cell suspensions were irradiated at ice temperature with 60Co gamma rays. Dose-response curves under aerobic and hypoxic conditions showed a much depressed base damage formation under hypoxia, which was created by blowing a stream of nitrogen across the cell suspensions for 30 min on ice. The presence of misonidazole had little or no detectable effect under hypoxia. It is concluded that an effect on the level of formation of thymine base damage is not primarily responsible for the radiosensitization by misonidazole under hypoxic conditions.     

Equilibrium dialysis studies of the binding of radioprotector compounds to DNA

Radioprotection by WR-2721, S-2-(3-aminopropylamino)ethyl phosphorothioate, is thought to involve its corresponding thiol (WR-1065) or symmetrical disulfide (WR-33278). It has been suggested that these metabolites concentrate close to the DNA target molecule; to test this hypothesis we have measured their in vitro binding to DNA. The binding of WR-33278 (0.05-0.4 mM) to calf thymus DNA (6 mM, with respect to DNA phosphate) was determined at 50, 100, and 150 mM KCl in 1 mM Tris, pH 7, by equilibrium dialysis. The binding of WR-1065 (0.5-8mM) was determined at 25, 50, and 100 mM KCl, under similar conditions, but with 2 mM EDTA and 3 mM dithiothreitol (DTT) added to the dialysis buffer to prevent thiol oxidation. Drug levels were quantitated by HPLC after fluorescent labeling with monobromobimane; disulfide samples were reduced with DTT prior to analysis. Dissociation constants (Kd = [Free Drug] [DNA site]/ [bound drug] ) under these conditions were found to vary with ionic strength, being in the range of 0.02 +/- 0.01 to 0.18 +/- 0.06 mM for WR-33278 and 0.43 +/- 0.24 to 3.5 +2/- 1.5 mM for WR-1065. WR-2721, glutathione, cysteine, and DTT showed no detectable binding to DNA in 25 mM KCl. However, cysteamine and cystamine did bind to DNA, with unbound drug to bound drug ratios of 8 +/- 2 and 0.6 +/- 0.1, respectively, at total drug concentrations of 1 mM. Cystamine and WR-1065 bound to DNA with comparable affinity under similar conditions. These results indicate that binding of WR-33278 and WR-1065 by DNA phosphate are probably significant in the mechanism of radioprotection by WR-2721.     

Evaluation of various types of new hypoxic cell sensitizers using the EMT6 single cell-spheroid-solid tumour system

Eleven new hypoxic cell sensitizers representative of those developed in Japan between 1980 and 1985 were evaluated in vitro and in vivo in comparison with misonidazole (MISO), SR-2508, Ro 03-8799, and ANT (2-amino-5-nitrothiazole). The new compounds included 2-nitroimidazole nucleoside analogues, nitrotriazoles and other nitroaromatics, non-nitro compounds, and electron-affinic compounds that readily intercalate DNA. The sensitizing activity in the EMT6 single cells correlated not only with the reduction potential but, for some compounds, also with the reactivity with non-protein sulphydryls. The sensitizers were also tested using the EMT6 spheroids and solid tumours. The patterns of changes in sensitizer enhancement ratios (SERs) for single cells, spheroids, and solid tumours were classified into two types: (1) SERs for the three testing systems were similar; and (2) SERs decreased in the order of: single cells, spheroids, and solid tumours. Only nitroimidazole and nitrotriazole derivatives belonged to the former type. RK-28 and RK-29, 2-nitroimidazoles with sugar analogue components, had in vivo effects almost equal to those of MISO. Also 3- and 4-nitrotriazole derivatives had definite in vivo effects.