Aerobic radioprotection of pBR322 by thiols: effect of thiol net charge upon scavenging of hydroxyl radicals and repair of DNA radicals.

The extent of conversion of supercoiled pBR322 plasmid DNA to the open circular and linear forms can be measured by HPLC on a Waters Gen Pak FAX column following in vitro gamma irradiation of the DNA. This radiation effect has proven to be useful for the study of the radioprotection of DNA by thiols and other drugs. This system was used with gamma irradiation in air at pH 7.0 and physiological ionic strength to compare radioprotection by a series of thiols, disulfides, and thioethers, all having approximately 10(8) s-1 effective hydroxyl radical scavenging rate (10 mm dm-3 drug) and having net charge (Z) ranging from -2 to +3. All sulfur compounds exhibited substantial protection due to scavenging of hydroxyl radicals in bulk solution but thiols exhibited a 24-fold variation in relative ability to protect the plasmid DNA from strand breaks, as assessed from the dose-response curves: mercaptosuccinate (Z = -2), 0.53; GSH (Z = -1), 0.67; 3-mercaptopropionate (Z = -1) 0.80; mercaptoethanol (Z = 0), 1.00; dithiothreitol (Z = 0), 1.5; cysteamine (Z = +1), 3.7; N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, Z = +2), 6.7; N1-(2-mercaptoethyl)spermidine (WR-35980, Z = +3), 12. Comparison of these results with those obtained using disulfide and thioether radioprotectors indicated that local scavenging of hydroxyl radicals near DNA increases slightly with Z, apparently as a result of variations in thiol concentration near DNA, but this accounts for only a small fraction of the change with Z found for cationic thiols. The marked increase in protection found for cationic thiols was attributed to chemical repair of DNA radicals and was in accord with predictions based upon recently measured rates for chemical repair of DNA radicals and was in accord with predictions based upon recently measured rates for chemical repair of pBR322 radicals. It is concluded that chemical repair of DNA radicals by anionic thiols does not compete with the oxygen fixation reaction in air and that protection by these thiols occurs primarily via the scavenging of hydroxyl radicals. However, chemical repair of DNA radicals is significantly enhanced by counterion condensation for cationic thiols and becomes a significant factor in their ability to protect DNA against radiation damage under aerobic conditions.     

Interaction of glutathione and other low-molecular-weight thiols with DNA: evidence for counterion condensation and coion depletion near DNA

The interaction of low-molecular-weight thiols with sonicated DNA was examined using spin filtration to concentrate the DNA. Cationic thiols (WR 1065 and cysteamine) behaved as counterions and were found to have increased concentrations in the DNA retentate relative to the filtrate. Anionic thiols (GSH, 2-mercaptoethanesulfonate, mercaptosuccinate) behaved as coions and were decreased in concentration in the DNA fraction. Concentrations of the uncharged thiol 2-mercaptoethanol were little influenced by DNA. The results demonstrate the importance of counterion condensation and coion depletion in determining the concentrations of charged species near DNA. They provide a rationale for enhanced effectiveness of WR 1065 and cysteamine as radioprotectors compared to neutral and anionic thiols and suggest that anionic thiols such as GSH should be poor radioprotectors of DNA.     

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.     

Enhancement of topoisomerase I-mediated unwinding of supercoiled DNA by the radioprotector WR-33278

The radioprotector WR-33278, the disulfide of WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane), is shown to stimulate eukaryotic topoisomerase I unwinding of negatively supercoiled DNA. This observation suggests the possibility that some protection may be conferred to DNA either by a decrease in its supercoiled state or by altering directly other enzymatic processes. This is the first report of a radioprotective compound stimulating an enzyme involved in DNA structure and synthesis.     

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.     

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.     

Effect of radioprotectors on lipid peroxidation in liver microsomes induced by ultraviolet irradiation of the skin in rats

The modifying effect of radioprotectors (serotonin, cysteamine, ionol) on lipid peroxidation intensification of liver microsomes caused by rat skin ultraviolet radiation has been studied. A possible mechanism of action of these compounds on the investigated indexes during their preventive injection is under discussion.     

Induction of marrow hypoxia by radioprotective agents

The ability of thiol and non-thiol radioprotectors to induce hypoxia was determined using the binding of [3H]misonidazole by bone marrow cells as a measure of hypoxia. When administered at maximally radioprotective doses, four drugs (WR-2721, cysteamine, 5-hydroxytryptamine, and 16,16-dimethyl prostaglandin E2) significantly increased the amount of [3H]misonidazole bound by marrow cells, while no significant increase in binding was observed with three other agents (endotoxin, AET, superoxide dimutase). Doses of WR-2721 previously shown to provide suboptimal radioprotection did not significantly increase 3H-misonidazole binding. These results suggest that the physiological effects of some radioprotectors, that is, their ability to induce marrow hypoxia, may contribute to their efficacy in vivo.     

Radioprotection of DNA by thiols: relationship between the net charge on a thiol and its ability to protect DNA

Release of free bases from calf thymus DNA upon irradiation in aerated 0.1 mol dm-3NaClO4 at pH 7 has been measured by HPLC and shown to be markedly influenced by the presence of thiols during irradiation. The ability of thiols to protect DNA was shown to depend upon the net charge (Z) at pH 7 in the order WR 1065 (Z = +2) greater than cysteamine (Z = +1) greater than 2-mercaptoethanol (Z = 0) approximately equal to dithiothreitol (Z = 0) greater than GSH (Z = -1) approximately equal to 2-mercaptoethanesulfonic acid (Z = -1) approximately equal to 2-mercaptosuccinate (Z = -2). A similar dependence of protection upon net charge was found for disulfides: cystamine (Z = +2) greater than 2-mercaptoethyl disulfide (Z = 0) greater than GSSG (Z = -2). Protection by WR 1065, but not by 2-mercaptoethanol or GSH, was found to decrease significantly with increasing ionic strength. Protection by WR 1065 and GSH was not markedly dependent upon pH between pH 6 and 8. The results are explained in terms of electrostatic interaction of the thiols with DNA, leading to high concentrations of cations near DNA, which allow them to scavenge hydroxyl radicals and repair DNA radicals effectively and to low concentrations of anionic thiols near DNA, which limit their effectiveness as protectors. Poly(dG,dC) and calf thymus DNA exhibited comparable release of G and C upon changing from 0.1 to 0.7 mol dm-3 MgSO4. Since this change causes poly(dG,dC), but not calf thymus DNA, to undergo a change from the B-form to the Z-form of DNA, both forms must have a comparable susceptibility to radiation-induced base release.     

Different effects of trifluoroethanol and glycerol on the stability of tropomyosin helices and the head-to-tail complex

Tropomyosin (Tm) is a dimeric coiled-coil protein, composed of 284 amino acids (410 A), that forms linear homopolymers through head-to-tail interactions at low ionic strength. The head-to-tail complex involves the overlap of approximately nine N-terminal residues of one molecule with nine C-terminal residues of another Tm molecule. In this study, we investigate the influence of 2,2,2-trifluoroethanol (TFE) and glycerol on the stability of recombinant Tm fragments (ASTm1-142, Tm143-284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm143-284(5OHW269)) contains a 5-hydroxytryptophan (5OHW) probe at position 269 whose fluorescence is sensitive to the head-to-tail interaction and allows us to accompany titrations of Tm143-284(5OHW269) with ASTm1-142 to calculate the dissociation constant (Kd) and the interaction energy at TFE and glycerol concentrations between 0% and 15%. We observe that TFE, but not glycerol, reduces the stability of the head-to-tail complex. Thermal denaturation experiments also showed that the head-to-tail complex increases the overall conformational stability of the Tm fragments. Urea and thermal denaturation assays demonstrated that both TFE and glycerol increase the stability of the isolated N- and C-terminal fragments; however, only TFE caused a significant reduction in the cooperativity of unfolding these fragments. Our results show that these two cosolvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their opposing effects on head-to-tail complex formation.     

Tropomyosin exon 6b is troponin-specific and required for correct acto-myosin regulation

The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT(1)-tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 microm) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin.Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin.Tm.Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin.Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.     

A comparative study of the antimutagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster

The 1,4-dihydropyridine derivative 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (1,4-DHP) was studied for antimutagenic effects in the dominant lethal test and in the sex-linked recessive lethal test of Drosophila melanogaster. The observed effects were compared with those of the radioprotectors cysteine and cysteamine and with those of the phenolic antioxidant butylated hydroxytoluene (BHT). In a wide range of concentrations, including low ones, 1,4-DHP reduces the frequency of EMS-induced genetic damage (point mutations and chromosome breakage). A reduction of the mutation rate induced by EMS in adults could be observed independently of the developmental stages (larvae or imago) pretreated with 1,4-DHP. The protective effect of this new antimutagen against the alkylating agent depended on both the 1,4-DHP dose and the level of the EMS-induced mutation rate. The effect of 1,4-DHP was more pronounced than that of the studied radioprotectors. It is concluded that dihydropyridine-type compounds are able to protect eukaryote germs cells from genetic damage produced by direct-acting mutagens such as EMS.     

The effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and X-irradiated Escherichia coli

In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to X rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between X-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I-, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.     

Influence of hydrogen bonding in DNA and polynucleotides on reaction of nitrogens and oxygens toward ethylnitrosourea.

The reactivity of ethylnitrosourea toward hydrogen-bonded sites in double-stranded DNA or oly(rA).poly(rU) was compared with those sites in single-stranded DNA, RNA, or poly(rA). Alkylation of the N-1 of A in poly(rA).poly(rU) was almost suppressed at 5 degrees C but could be markedly increased by raining the reaction temperature to 25 degrees C, well below the Tm of 56 degrees C. In contrast, the N-7 and N-6 of A, which are not hydrogen bonded, reacted to the same extent at temperatures ranging from 5 to 65 degrees C. The extent of reaction at the N-3 of A varied inversely with the reactivity of the N-1 of A, indicating that of these two nitrogens the N-1 of A is the most reactive. The proportion of reaction at the various nitrogens in poly(rA) was not affected by temperature. Hydrogen-bonded oxygens in double-stranded DNA are the O-6 of G, the O-4 of T, and the O2 of C. All are equally reactive at 5, 25, and 51 degrees C. It is concluded that the observed temperature independence is due to these oxygens having an electron pair not involved in hydrogen bonding and, thus, available for reaction. In contrast, the electron pair of the N-1 of A (or the N-3 of C) is involved in hydrogen bonding, and the extent of their reactivity is dependent on thermal fluctuation providing transiently open base pairs at temperatures far below the Tm.     

23Na NMR study of DNA thermal transconformation in presence of cysteamine radioprotector

DNA thermal transconformation is studied in absence and in presence of the cysteamine radioprotector, by observing the delta nu 1/2 variation of 23Na NMR peaks. The sodium state (Free or Bound) is discussed with the help of a two states model with RF and RB relaxation rates. The delta nu 1/2 behaviour during the DNA transconformation shows clearly the electrostatic interaction with cysteamine which is accompanied by an Na+ ejection out of phosphate sites. The temperature dependence of delta nu 1/2 in all cases leads to the conclusion that RBc (the average relaxation rate of sodium nuclei that remain bound in the coil state of DNA) tends to zero.     

Modulation of bleomycin-induced mitotic recombination in yeast by the aminothiols cysteamine and WR-1065

The cancer chemotherapy drug bleomycin (BLM) is a potent inducer of genetic damage in a wide variety of assays. The radioprotectors cysteamine (CSM) and WR-1065 have been shown in previous studies to potentiate the induction of micronuclei and chromosome aberrations by BLM in Go human lymphocytes. By contrast, WR-1065 is reported to reduce the induction of hprt mutations by BLM in Chinese hamster cells. To elucidate the basis for these interactions, we examined the effects of CSM and WR-1065 on the induction of mitotic gene conversion by BLM in the yeast Saccharomyces cerevisiae. Treatment with BLM causes a dose-dependent increase in the frequency of mitotic gene conversion and gene mutations. Unlike its potentiation of BLM in G₀ lymphocytes, WR-1065 protected against the recombinagenicity of BLM in yeast. CSM was also strongly antirecombinagenic under some conditions., but the nature of the interaction depended strongly on the treatment conditions. Under hypoxic conditions, cysteamine protected against BLM, but under oxygenrich conditions CSM potentiated the genetic activity og BLM. The protective effect of aminothiols against BLM may be ascribed to the depletion of oxygen required for the activation of BLM and the processing of BLM-induced damage. Aminothiols may potentiatc the effect of BLM by acting as an electron source for the activation of BLM and/or by causing conformational alterations that make DNA more accessible tc BLM. The results indicate that aminothiols have a strong modulating influence on the genotoxicity of BLM in yeast as they do in other genetic assays. Moreover, the modulation differs markedly depending on physiological conditions. Thus, yeast assays help to explain why aminothiols have been observed to potentiate BLM in some genetic systems and to protect against it in others.     

Action of modifiers of a radiation lesion on the binding of prostaglandin E2 with liver membranes in F1 CBA X C57BL strain mice

Prostaglandin E2 (PGE2) was specifically bound by the membrane fraction prepared from the mouse liver. The binding constants indicate the presence of high-affinity PGE2 binding sites with an apparent dissociation constant (Kd) of 0.82 X 10(-9) M and a capacity of 0.36 X 10(9) M/mg protein and a lower affinity PGE2 binding site with Kd = 15.73 X 10(-9) M and a capacity of 5.31 X 10(9) M/mg protein. The radioprotectors, MEA and APAETP inhibit PGE2 binding and alter its kinetics. Apparently the mechanism of PGE2 binding by membranes is related to interaction of prostaglandins with thiols and sufhydryl groups of membrane lipoproteins, while the radioprotectors modify the functional groups participating in receptor PGE2 binding.     

Thermal unfolding of smooth muscle and nonmuscle tropomyosin alpha-homodimers with alternatively spliced exons

We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.     

Studies on interaction between poly(L-lysine58, L-phenylalanine42) and deoxyribonucleic acids.

A random copolymer of 58% L-lysine and 42% L-phenylalanine, poly(Lys58Phe42), was used as a model protein for studying the role of phenylalanine residues in protein-DNA interaction. Complexes between this copolypeptide and DNA, made by direct mixing, were studied by absorbance, circular dichroism (CD), fluorescence, and thermal denaturation. Complex formation results in an increase in absorbance, and an enhancement, red-shift, and broadening of phenylalanine fluorescence. The fluorescence enhancement is opposite to the quenching observed when a tyrosine copolypeptide is bound to DNA (R. M. Santella and H.J. Li (1974), Biopolymers 13, 1909). The positive CD band of DNA near 275 nm is reduced and red-shifted by the binding of the phenylalanine copolypeptide to a greater extent than by the tyrosine copolypeptide. Thermal denaturation of the complexes in 2.5 times 10(-4) M EDTA (pH 8.0) shows three characteristic melting bands. For complexes with calf thymus DNA, free base pairs melt at Tm,I (47-49 degrees) and copolypeptide-bound base pairs show two melting bands (Tm,II at 73-75 degrees, and Tm,III at 88 -90 degrees). Similar thermal denaturation results have been observed for complexes with Micrococcus luteus DNA. The fluorecence intensity of the complexes is greatly increased when the temperature is raised to the Tm,II region. In addition to fluorescence measurements, the effects of increasing temperature on absorption and CD spectra of the complexes were also studied. Stacking interaction between the phenylalanine chromophore and DNA bases, either partial or full intercalation, is implicated by the experimental results. Several mechanisms are proposed to describe the reaction between the copolypeptide and DNA, and thermal denaturation of the complex.