The disposition of ethiofos (WR-2721) in the isolated perfused rat liver

We have investigated the disposition of ethiofos (20 mg, 4 microCi [14C]ethiofos) in the isolated perfused rat liver preparation to determine the hepatic contribution to the poor oral bioavailability of the drug. Ethiofos clearance (10.6 +/- 3.3 ml h-1) was only a small fraction (1.2 +/- 0.03%) of the perfusate flow rate. The elimination half-life was calculated at 7.1 +/- 1.9 h. The area under curve, AUC0-4 h, for ethiofos (2858 +/- 314 nM h ml-1) was not significantly different from that of 14C (3038 +/- 692 nM h ml-1) or total material convertible to WR-1065 (total WR-1065, 3324 +/- 612 nM h ml-1), indicating a low level of metabolism. The AUC0-4 h for free WR-1065 (37.5 +/- 23.3 nM h ml-1) was less than 2% of ethiofos. Biliary elimination of ethiofos, WR-1065, and 14C was below 1%. At 4 h postdose, 7.9 +/- 1.9% of the dose of radioactivity remained in the liver. Less than 1.5% could be identified as ethiofos (0.12 +/- 0.09%) or total WR-1065 (1.09 +/- 0.05%). Ethiofos, 14C, and total WR-1065 were approximately evenly distributed between the 10,000-g pellet and supernatant. However, significantly more ethiofos, WR-1065, and 14C were recovered from the 105,000-g supernatant compared with the pellet. In summary, both the metabolism and biliary elimination of ethiofos and its derivatives were sparing. Hence it is likely that in the rat, the contribution of the liver to the presystemic biotransformation and poor bioavailability of ethiofos is relatively minor.     

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.     

Thiol uptake by Chinese hamster V79 cells and aerobic radioprotection as a function of the net charge on the thiol.

A series of thiols having net charge (Z) varying from -2 to +3 were studied using aerobic suspensions of Chinese hamster V79-171 cells in pH 7.4 medium at 297 K to evaluate the rate of uptake by cells and the extent of radioprotection as a function of thiol concentration in cells. For measurement of cellular levels, cells were separated from medium by centrifugation through silicone oil and tritiated water was employed to determine cell water volume. Estimated half-lives for uptake were: 2-mercaptosuccinate (Z = -2), greater than or equal to 1 h; 3-mercaptopropanoate (MPA, Z = -1), less than 2 min; 2-mercaptoethanol (2ME, Z = 0), less than 2 min; cysteamine (CyA, Z = +1), less than 2 min; N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, Z approximately +2), approximately 40 min; N1-(2-mercaptoethyl)spermidine (WR-35980, Z approximately +3), greater than or equal to 10 h. After equilibration the cellular concentration of MPA was 60 +/- 8% of the medium level; the corresponding values for 2ME and CyA were 95 +/- 3 and 180 +/- 12%, respectively, but equilibrium was not reached for the other thiols studied. Those thiols taken up at significant rates were evaluated in terms of their ability to protect against aerobic gamma-ray-induced lethality. The results, summarized in terms of the cellular concentration of thiol (mmol dm-3) needed to achieve an aerobic radioprotection factor of 1.5, were as follows: MPA, 80 +/- 15; 2ME, 24 +/- 2; CyA, 4.7 +/- 1.3; WR-1065, 3.4 +/- 0.6. These values accorded well with those predicted from hydroxyl radical scavenging and DNA radical repair rates obtained using pBR322 DNA as a model system. This shows that hydroxyl radical scavenging and DNA radical repair are important mechanisms in the protection of cells by thiols and that the net charge on the thiol is a significant factor in its effectiveness. The results indicate that in air hydroxyl radical scavenging is the dominant mode of action by MPA, but that chemical repair of DNA radicals becomes significant for 2ME and is the dominant mechanism of protection for CyA and WR-1065.     

Radioprotective thiolamines WR-1065 and WR-33278 selectively denature nonhistone nuclear proteins

Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 degrees C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 degrees C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR-1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca(2+)ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.     

Determination of WR-1065 in human blood by high-performance liquid chromatography following fluorescent derivatization by a maleimide reagent ThioGlo3

In order to improve the sensitivity and stability of human blood samples containing WR-1065 (i.e., active metabolite of the cytoprotective agent amifostine), a high-performance liquid chromatographic method was developed and validated using fluorescent derivatization with ThioGlo3. Using a sample volume of only 100 microl, the method was specific, sensitive (limit of quantitation=10 nM in deproteinized blood or 20 nM in whole blood), accurate (error < or = 3.2%) and reproducible (CV < or = 8.7%). In addition, the stability of WR-1065 in deproteinized and derivatized blood samples was assured for at least four weeks at -20 degrees C. This method should be particularly valuable in translating the kinetic-dynamic relationship of WR-1065 in preclinical models to that in cancer patients.     

Properties of selected S-nitrosothiols compared to nitrosylated WR-1065

WR-1065 ([N-mercaptoethyl]-1-3-diaminopropane), the active form of the aminothiol drug Ethyol/Amifostine, protects against toxicity caused by radiation, chemotherapy and endotoxin. Because WR-1065 and other thiols readily bind nitric oxide (NO), injurious conditions or therapies that induce the production or mobilization of NO could alter the effects of WR-1065. S-Nitrosothiols were prepared from various thiols by a standard method to compare properties and stability. Heteromolecular quantum correlation 2D nuclear magnetic resonance was used to characterize nitrosylated glutathione (GSH) and WR-1065; both S- and N-nitrosothiols were observed, depending on the experimental conditions. Three categories of S-nitrosothiol stability were observed: (1) highly stable, with t(1/2) > 8 h, N-acetyl-L-cysteine nitrosothiol (t(1/2) 15 h) > GSH nitrosothiol (t(1/2) 8 h); (2) intermediate stability, t(1/2) approximately 2 h, cysteamine nitrosothiol and WR-1065 nitrosothiol; and (3) low stability, t(1/2) < 1 h, cysteine nitrosothiol and Captopril nitrosothiol. Similar relative rates were observed for Hg(+2)-induced denitrosylation: WR-1065 reacted faster than GSH nitrosothiol, while GSH nitrosothiol reacted faster than N-acetyl-L-cysteine nitrosothiol. Mostly mediated by mixed-NPSH disulfide formation, the activity of the redox-sensitive cysteine protease, cathepsin H, was inhibited by the S-nitrosothiols, with WR-1065 nitrosothiol > cysteine nitrosothiol > N-acetyl-L-cysteine nitrosothiol and GSH nitrosothiol. These observations indicate that, relative to other nitrosylated non-protein thiols, the S-nitrosothiol of WR-1065 is an unstable non-protein S-nitrosothiols with a high reactive potential in the modification of protein thiols.     

Factors influencing the oxidation of the radioprotector WR-1065

N-(2-Mercaptoethyl)-1,3-diaminopropane (WR-1065) is the free thiol form of the radio- and chemoprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Interest currently exists in the clinical use of WR-2721 and WR-1065 as radio- and chemoprotectors of normal tissues. However, measurement of plasma levels of WR-1065 has proven difficult, due to rapid drug oxidation. Therefore, we studied factors influencing the oxidation of WR-1065, in Hepes-buffered saline as well as in tissue culture media containing 10% fetal bovine serum. The rate of oxygen consumption by WR-1065, as determined using the Clark oxygen electrode system, was faster in medium plus serum than in Hepes-buffered saline. That this effect is largely due to the presence of trace metal ions in tissue culture media and serum was indicated by the observation that addition of Cu2+ or Fe3+ to buffer stimulated oxygen consumption. Addition of KCN inhibited the reaction of WR-1065 with oxygen, and this effect was dependent on KCN concentration. That KCN blocked WR-1065 oxidation to the disulfide was verified using Ellman's reagent to quantitate the free thiol form. The rate of oxygen consumption was shown to be affected by temperature as well as concentration of WR-1065. Catalase reduced the rate of oxygen consumption of WR-1065, indicating that peroxide is formed in this system. Superoxide dismutase had a stimulatory effect. WR-1065 was found to stimulate the hexose monophosphate shunt in A549 cells. Since this stimulation was prevented by the presence of catalase, it appeared to be due to the response of the cells to peroxide, formed as a result of WR-1065 autooxidation.     

Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 μmol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%).     

Association of WR-1065 with CHO AA8 cells, nuclei, and nucleoids

The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.     

Effect of temperature on atrial and ventricular adenosine A1 receptors of the guinea pig

Cardiac adenosine receptors are coupled to adenylate cyclase inhibition. In the guinea pig heart, the relative agonist potencies observed for adenylate cyclase inhibition were R-N6-phenylisopropyladenosine (R-PIA) = N6-cyclohexyladenosine greater than 5'-N-ethylcarboxamidoadenosine much greater than S-PIA. In both atrial and ventricular membranes, the antagonists 8-phenyltheophylline (8-PT) and isobutylmethylxanthine (IBMX) also showed similar affinities for atrial and ventricular adenosine receptors. The same pattern of relative agonist potencies was observed in experiments performed at either 25 or 37 degrees C. However, the maximal inhibition produced by R-PIA in atrial membranes decreased from 30.8 +/- 3.2% (n = 7) at 25 degrees C to 18.8 +/- 1.6% (n = 4) at 37 degrees C. No such difference in maximal inhibition was observed with ventricular membranes at these two temperatures (34.5 +/- 1.6%, n = 6 at 25 degrees C and 35.3 +/- 0.9%, n = 11 at 37 degrees C). While there was no change in agonist potencies, the affinities of the antagonists 8-PT and IBMX at cardiac adenosine A1 receptors were affected by temperature. At 25 degrees C, the pKD values for 8-PT and IBMX in ventricular membranes were 4.65 +/- 0.21 (n = 3) and 4.55 +/- 0.20 (n = 3), respectively. Their affinities were 7- to 19-fold higher at 37 degrees C, the pKD values being 5.93 +/- 0.12 (n = 7) (p less than 0.02) and 5.38 +/- 0.18 (n = 3) (p less than 0.05), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death.

Gamma-irradiation, glucocorticoid hormones, and calcium ionophores stimulate a suicide process in thymocytes, known as apoptosis or programmed cell death, that involves internucleosomal DNA fragmentation by a Ca(2+)- and Mg(2+)-dependent nuclear endonuclease. In this study we report that N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) blocked DNA fragmentation and cell death in thymocytes exposed to gamma-radiation, dexamethasone, or calcium ionophore A23187. WR-1065 protected the thymocytes from radiation-induced apoptosis when incubated with cells after irradiation but not before and/or during irradiation. WR-1065 inhibited Ca(2+)- and Mg(2+)-dependent DNA fragmentation in isolated thymocyte nuclei. Our results suggest that WR-1065 protects thymocytes from apoptosis by inhibiting Ca(2+)- and Mg(2+)-dependent nuclear endonuclease action.     

The radioprotective agent, amifostine, suppresses the reactivity of intralysosomal iron

Amifostine (2-[(3-aminopropyl)amino]ethane-thiol dihydrogen phosphate ester; WR-2721) is a radioprotective agent used clinically to minimize damage from radiation therapy to adjacent normal tissues. This inorganic thiophosphate requires dephosphorylation to produce the active, cell-permeant thiol metabolite, WR-1065. The activation step is presumably catalyzed by membrane-bound alkaline phosphatase, activity of which is substantially higher in the endothelium of normal tissues. This site-specific delivery may explain the preferential protection of normal versus neoplastic tissues. Although it was developed several decades ago, the mechanisms through which this agent exerts its protective effects remain unknown. Because WR-1065 is a weak base (pKa = 9.2), we hypothesized that the drug should preferentially accumulate (via proton trapping) within the acidic environment of intracellular lysosomes. These organelles contain abundant 'loose' iron and represent a likely initial target for oxidant- and radiation-mediated damage. We further hypothesized that, within the lysosomal compartment, the thiol groups of WR-1065 would interact with this iron, thereby minimizing iron-catalyzed lysosomal damage and ensuing cell death. A similar mechanism of protection via intralysosomal iron chelation has been invoked for the hexadentate iron chelator, desferrioxamine (DFO; although DFO enters the lysosomal compartment by endocytosis, not proton trapping). Using cultured J774 cells as a model system, we found substantial accumulation of WR-1065 within intracellular granules as revealed by reaction with the thiol-binding fluorochrome, BODIPY FL L-cystine. These granules are lysosomes as indicated by co-localization of BODIPY staining with LysoTracker Red. Compared to 1 mM DFO, cells pre-treated with 0.4 microM WR-1065 are protected from hydrogen peroxide-mediated lysosomal rupture and ensuing cell death. On a molar basis in this experimental system, WR-1065 is approximately 2500 times more effective than DFO in preventing oxidant-induced lysosomal rupture and cell death. This increased effectiveness is most likely due to the preferential concentration of this weak base within the acidic lysosomal apparatus. By electron spin resonance, we found that the generation of hydroxyl radical, which normally occurs following addition of hydrogen peroxide to J774 cells, is totally blocked by pretreatment with either WR-1065 or DFO. These findings suggest a single and plausible explanation for the radioprotective effects of amifostine and may provide a basis for the design of even more effective radio- and chemoprotective drugs.     

Manganese superoxide dismutase (SOD2)-mediated delayed radioprotection induced by the free thiol form of amifostine and tumor necrosis factor alpha

RKO36 cells, a subclone of RKO colorectal carcinoma cells that have been stably transfected with the pCMV-EGFP2Xho vector, were grown to confluence and then exposed to either the radioprotector WR-1065, i.e. the active thiol form of amifostine, for 30 min at doses of 40 microM and 4 mM or the cytokine tumor necrosis factor alpha (TNFalpha, TNFA) for 30 min at a concentration of 10 ng/ml and then washed. Total protein was isolated as a function of time up to 32 h after these treatments. Both doses of WR-1065 as well as the concentration of TNFalpha used were effective in elevating intracellular levels of the antioxidant protein SOD2 (also known as MnSOD) at least 15-fold over background levels as determined by Western blot analysis, while measured SOD2 activity was elevated between 5.5- and 6.9-fold. SOD2 reached a maximal level 24 h and 20 h after WR-1065 and TNFalpha treatments, respectively. The antioxidant proteins catalase and glutathione peroxidase (GPX) were also monitored over the 32-h period. In contrast to the robust changes observed in intracellular levels of SOD2 as a function of time after exposure of cells to WR-1065, catalase levels were elevated only 2.6-fold over background as determined by Western blot analysis, while GPX activity was unaffected by WR-1065 exposure. GPX protein levels were extremely low in cells, and analysis of GPX activity using a spectrophotometric method based on the consumption of reduced NADPH also revealed no measurable change as a function of WR-1065 or TNFalpha exposure. RKO36 cells either were irradiated with X rays in the presence of either 40 microM or 4 mM WR-1065 or 10 ng/ml TNFalpha or were irradiated 24 or 20 h later, respectively, when SOD2 protein levels were most elevated. The concentrations and exposure conditions used for WR-1065 and TNFalpha were not cytotoxic and had no effect on plating efficiencies or cell survival compared to untreated controls. No protection or sensitization was observed for cells irradiated in the presence of 40 microM WR-1065 or TNFalpha. Survival was elevated 1.90-fold for cells irradiated in the presence of 4 mM WR-1065. When RKO36 cells were irradiated with 2 Gy 24 h after 40 microM or 4 mM WR-1065 and 20 h after TNFalpha treatments when SOD2 levels were the most increased, survival was elevated 1.42-, 1.48- and 1.36-fold, respectively. This increased survival represents a SOD2-mediated delayed radioprotective effect. SOD2 appears to be an important antioxidant gene whose inducible expression is an important element in adaptive cellular responses in general, and the delayed radioprotective effect in particular. It can be induced by a range of agents including cytoprotective nonprotein thiols such as WR-1065 and pleiotropic cytokines such as TNFalpha.     

Glucose-6-phosphate dehydrogenase from brewers' yeast. The effects of pH and temperature on the steady-state kinetic parameters of the two-chain protein species.

A systematic study has been made of the pH- and temperature-dependency of the steady-state kinetic parameters of the stabilized two-subunit enzyme species of glucose-6-phosphate dehydrogenase, in the absence of superimposed association-dissociation reactions. The Vmax(app) data obtained in several buffers between pH 5 and 10 and at 18-32 degrees C lead to the postulate that at least two sets of protonic equilibria may govern the catalysis (one near pH 5.7 AT 25 DEGREES C and another near pH 9.2); furthermore, two pathways for product formation (i.e., two Vmax's) appear to be required to explain the biphasic nature of the log Vmax(app) vs. pH curves, with Vmax(basic) greater than Vmax(acidic + neutral). Of the several buffers explored, either a uniform degree of interaction or a minimal degree of buffer species interaction could be assessed from the enthalpy changes associated with the derived values for ionization constants attributed to the protonic equilibria in the enzyme-substrates ternary complexes for the case of Tris-acetate-EDTA buffers, at constant ionic strength. With the selection of this buffer at 0.1 (T/2) and at 25 and 32 degrees C, a self-consistent kinetic mechanism has emerged which allows for the random binding of the two fully ionized substrates to the enzyme via two major pathways, and product formation by both E-A--B- and HE-A--B-. As before (Kuby et al. Arch. Biochem, Biophys. 165, 153-178, 1974), a quasi-equilibrium is presumed, with rate-limiting steps (k + 5 and k + 5') at the interconversion of the ternary complexes. Values for the two sets of protonic equilibria defined by this mechanism (viz., pKk, pKH2 for the first ionizations, and pKk', pKH' for the second) could then be estimated. From their numerical values (e.g., at 25 degrees C: pKK = 5.7 PKH2 = 5.2; and pKK' = 9.1, PKH' = 8.2) and from the values for delta H degrees ioniz (e.g., delta H degrees pKK APPROXIMATELY 5.1 KCAL/MOL; DELTA H degrees pKK' APPROXIMATELY 11 KCAL/MOL), A POSTULATE IS PRESENTED WHICH ATTRIBUTES THESE Acid dissociation constants to an imidazole and epsilon-amino group, respectively.     

Accumulation of CHO cells in G2 phase following exposure to WR-1065

The radioprotector WR-1065 (2-[(aminopropyl)amino]ethanethiol) is known to protect mammalian cells from the cytotoxic and mutagenic effects of radio- and chemotherapeutic agents, but the exact mechanisms involved in this protection are not fully known. To help determine the effects of WR-1065 alone on cells, we examined its effect on a variety of cellular processes. Incubation of AA8 cells in 4 mM WR-1065 did not significantly affect the rate of DNA synthesis. Autoradiographic analysis of heavily labeled (S-phase population) nuclei of AA8 cells showed no significant difference in the S-phase population of WR-1065-treated versus control cells for up to 3 h. An examination of the effect of WR-1065 on repair synthesis, as measured by unscheduled DNA synthesis (UDS) in cells exposed to 15 Gy, showed no difference between treated and sham-treated cells for up to 2 h exposure. A significant reduction in the amount of UDS was seen in cells treated with the protector for 2.5 and 3 h. Incubation of cells in WR-1065 did alter the cell cycle distributions. An increase in the G2-phase population with a corresponding decrease in the G1-phase population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065. After the removal of WR-1065 at 3 h, a redistribution of the cells throughout the cell cycle occurred as has been observed in cells treated with other synchronization agents. These data suggest that perturbations in cell cycle progression, rather than direct effects on the rate of DNA synthesis, could play a role in the increased survival and reduced mutation frequencies observed in the presence of WR-1065.     

Opposite effects of WR-2721 and WR-1065 on radiation-induced hypothermia: possible correlation with oxygen uptake

Ionizing radiation induces hypothermia in guinea pigs. While systemic injection of the radioprotectant S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) did not block hyperthermia induced by exposure to 10 Gy of gamma radiation, central administration did attenuate it. The dephosphorylated metabolite of WR-2721, N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065), accentuated radiation-induced hypothermia by both routes of administration. In brain homogenates, oxygen uptake was inhibited by WR-2721 but elevated by WR-1065. These results suggest that the antagonism of radiation-induced hypothermia found only after central administration of WR-2721 is due to its direct actions and not to its dephosphorylated metabolite and that this effect may be correlated with the inhibition by WR-2721 of oxygen uptake.     

The radioprotective agent,amifostine, suppresses the reactivity of intralysosomal iron

Abstract

Amifostine (2-[(3-aminopropyl)amino]ethane-thiol dihydrogen phosphate ester; WR-2721) is a radioprotective agent used clinically to minimize damage from radiation therapy to adjacent normal tissues. This inorganic thiophosphate requires dephosphorylation to produce the active, cell-permeant thiol metabolite, WR-1065. The activation step is presumably catalyzed by membrane-bound alkaline phosphatase, activity of which is substantially higher in the endothelium of normal tissues. This site-specific delivery may explain the preferential protection of normal versus neoplastic tissues. Although it was developed several decades ago, the mechanisms through which this agent exerts its protective effects remain unknown. Because WR-1065 is a weak base (pKa = 9.2), we hypothesized that the drug should preferentially accumulate (via proton trapping) within the acidic environment of intracellular lysosomes. These organelles contain abundant 'loose' iron and represent a likely initial target for oxidant- and radiation-mediated damage. We further hypothesized that, within the lysosomal compartment, the thiol groups of WR-1065 would interact with this iron, thereby minimizing iron-catalyzed lysosomal damage and ensuing cell death. A similar mechanism of protection via intralysosomal iron chelation has been invoked for the hexadentate iron chelator, desferrioxamine (DFO; although DFO enters the lysosomal compartment by endocytosis, not proton trapping). Using cultured J774 cells as a model system, we found substantial accumulation of WR-1065 within intracellular granules as revealed by reaction with the thiol-binding fluorochrome, BODIPY FL L-cystine. These granules are lysosomes as indicated by co-localization of BODIPY staining with LysoTracker Red. Compared to 1 mM DFO, cells pre-treated with 0.4 μM WR-1065 are protected from hydrogen peroxide-mediated lysosomal rupture and ensuing cell death. On a molar basis in this experimental system, WR-1065 is approximately 2500 times more effective than DFO in preventing oxidant-induced lysosomal rupture and cell death. This increased effectiveness is most likely due to the preferential concentration of this weak base within the acidic lysosomal apparatus. By electron spin resonance, we found that the generation of hydroxyl radical, which normally occurs following addition of hydrogen peroxide to J774 cells, is totally blocked by pretreatment with either WR-1065 or DFO. These findings suggest a single and plausible explanation for the radioprotective effects of amifostine and may provide a basis for the design of even more effective radio- and chemoprotective drugs.     

Monensin-mediated transports of H+, Na+, K+ and Li+ ions across vesicular membranes: T-jump studies.

Theoretical expression for the rate of decay of delta pH across vesicular membrane due to carrier-mediated ion transports, 1/tau, has been modified taking note of carrier states (such as mon- and mon-H-M+) for which the translocation rate constants in the membrane are small. The rates of delta pH decay due to monensin-mediated H+ and M+ transports (M+ = Na+, K+, Li+) observed in our experiments in the pH range 6-8, and [M+] range 50-250 mM at 25 degrees C have been analysed with the help of this expression. delta pH across soybean phospholipid vesicular membranes were created by temperature jump in our experiments. The following could be inferred from our studies. (a) At low pH (approximately 6) 1/tau in a medium of Na+ is greater than that in a medium of K+. In contrast with this, at higher pH (approximately 7.5) 1/tau is greater in a medium of K+. Such contradictory observations could be understood with the help of our equation and the parameters determined in this work. The relative concentrations of the rate-limiting species (mon-H, mon-K, and mon-Li at Ph approximately 7 in vesicle solutions having Na+, K+ and Li+, respectively) can explain such behaviours. (b) The proton dissociation constant KH for mon-H in the lipid medium (pKH approximately 6.55) is larger than the reported KH in methanol. (c) The concentrations of mon- and mon-H-Na+ are not negligible under the conditions of our experiments. The latter species cause a [Na+]-dependent inhibition of ion transports. (d) The relative magnitudes of metal ion dissociation constants KHM (approximately 0.05 M) for mon-H-Na+ and KM (approximately 0.03 M) for mon-Na suggest that the carboxyl group involved in the protonation may not be dominantly involved in the metal ion complexation. (e) The estimates of KM (approximately 0.03 M for Na+, 0.5 M for K+ and 2.2 M for Li+) follow the ionophore selectivity order. (f) The rate constants k1 and k2 for the translocations of mon-H and mon-M (M+ = Na+, K+ and Li+) are similar in magnitude (approximately 9 x 10(3) s-1) and are higher than that for nig-H and nig-M (approximately 6 x 10(3) s-1) which can be expected from the relative molecular sizes of the ion carriers.     

Rates of dissociation of sex steroid hormones from human sex hormone-binding globulin: a reassessment

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure the rates of dissociation of dihydrotestosterone (DHT), testosterone (T), and estradiol (E2) from their binding proteins in human serum at 37 degrees C. Because measurements were begun after each hormone had fully (greater than 99%) dissociated from albumin, the observed rates of dissociation correspond to the rates of dissociation of the sex hormone-binding globulin (SHBG)-hormone complexes. The dissociation rate constants of the hormone-SHBG complexes were determined to be 0.016 +/- 0.001, 0.056 +/- 0.002, and 0.083 +/- 0.003 s-1 for DHT, T, and E2, respectively, corresponding to half-times of dissociation (t1/2) of 43, 12 and 8.4 s, respectively. The physiological significance of these findings can best be appreciated by comparing these t1/2 s with the capillary and sinusoidal transit times of various tissues (less than 1 s to approximately 10 s).