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L K Keefer

Publications and source records attributed to L K Keefer.

At least 55 records · Page 3Linked to original sources
PubMed1995

Nitric oxide and ethylnitrosourea: relative mutagenicity in the p53 tumor suppressor and hypoxanthine-phosphoribosyltransferase genes.

Nitric oxide (NO) is a cellular messenger which is mutagenic in bacteria and human TK6 cells and induces deamination of 5-methylcytosine (5meC) residues in vitro. The aims of this study were: (i) to investigate whether NO induces 5meC deamination in codon 248 of the p53 gene in cultured human bronchial epithelial cells (BEAS-2B); and (ii) to compare NO mutagenicity to that of ethylnitrosourea (ENU), a strong mutagen. Two approaches were used: (i) a novel genotypic assay, using RFLP/PCR technology on purified exon VII sequence of the p53 gene; and (ii) a phenotypic (HPRT) mutation assay using 6-thioguanine selection. BEAS-2B cells were either exposed to 4 mM DEA/NO (Et2N[N2O2]Na, an agent that spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation assay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU induces detectable numbers of G --> A transitions in codon 248 of p53 while 5-methylcytosine deamination was not detected in either iNOS-transfected cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose-responsively mutagenic in the phenotypic HPRT assay, reaching mutation frequencies of 24 and 96 times that of untreated control cells at ENU concentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO induced no detectable mutations in this assay, nor were any observed in cells transfected with murine iNOS. We conclude that if NO is at all promutagenic in these cells, it is significantly less so than the ethylating mutagen, ENU.

Base Sequence↗
PubMed1995

Nitric oxide suppression of human hematopoiesis in vitro. Contribution to inhibitory action of interferon-gamma and tumor necrosis factor-alpha.

IFN-gamma and TNF-alpha, potent inhibitors of hematopoiesis, induce nitric oxide synthase (NOS) in various cell types. When normal human bone marrow (BM) or CD34+ cells were exposed to NO, inhibition of colony formation was dose dependent and direct. NO induced apoptosis in BM progenitors, as shown by electrophoretic detection of DNA degradation and deoxynucleotidyl transferase assay. Using PCR and immunoprecipitation, we found inducible NOS (iNOS) mRNA and iNOS protein in BM after stimulation with IFN-gamma or TNF-alpha. iNOS mRNA was also detected by PCR in highly purified CD34+ cells; TNF-alpha or IFN-gamma increased iNOS expression. The presence of iNOS in CD34+ cells was confirmed in single cells by immunochemical staining. NG-Monomethyl-L-arginine (MM-Arg), an NOS inhibitor, partially reversed the effects of TNF-alpha and, to a lesser extent, IFN-gamma in methylcellulose culture of total BM and CD34+ cells, and inhibited apoptosis of BM cells induced by these cytokines. When the effects of competitive iNOS inhibition were tested on more immature progenitors, MM-Arg increased the number of long-term BM culture-initiating cells in control cultures but failed to protect these cells from the inhibitory action of IFN-gamma and TNF-alpha. Our results suggest that NO may be one mediator of cytokine-induced hematopoietic suppression.

Amino Acid Oxidoreductases↗
PubMed1995

Nitric oxide (NO) donor molecules: effect of NO release rate on vascular smooth muscle cell proliferation in vitro.

Nitric oxide (NO) inhibits vascular smooth muscle cell (SMC) growth in vitro. To determine the effects of release rate and exposure time on SMC growth inhibition by NO, we compared the activities of five NO donors that generate NO with half-lives of 2 min (DEA/NO, Et2N[N2O2]Na), 15 min (PAPA/NO, CH3(CH2)2N[N2O2]-(CH2)3NH3+), 39 min, (SPER/NO, H2N(CH2)3NH2+(CH2)4N[N2O2]-(CH2)3NH2), 3 h (DPTA/NO, H2N(CH2)3N[N2O2]-(CH2)3NH3+), and 20 h (DETA/NO, H2N(CH2)2N[N2O2]-(CH2)2NH3+). After 22-h treatment, rat aorta SMC (RA-SMC) DNA synthesis was inhibited with IC50 values of 180, 60, and 40 microM for SPER/NO, DPTA/NO, and DETA/NO, respectively. DEA/NO and PAPA/NO did not inhibit DNA synthesis significantly at any concentration tested (20-500 microM). The inhibitory effect of NO on RA-SMC DNA synthesis was thus greatest when a given molar dose of NO was delivered slowly throughout the 22-h period. The antiproliferative effect of DETA/NO was confirmed by measurement of cell numbers for 7 days. When RA-SMC were treated with 500 microM DETA/NO on days 1, 3, and 5, growth was completely suppressed. Cell viability was > 95%, confirming that DETA/NO was not cytotoxic. The results suggest that NO donors may be useful inhibitors of intimal hyperplasia and restenosis after vascular injury such as balloon angioplasty.

Angioplasty, Balloon↗
PubMed1994

In vivo radiation protection by nitric oxide modulation.

Drugs that affect blood flow have been shown to be whole body radiation protectors. Using NG-nitro-L-arginine, a specific inhibitor of nitric oxide synthase, and the NO-releasing agent (C2H5)2N[N(O)NO-]Na+ (DEA/NO), we have studied the ability of NO to modulate whole body radiation toxicity in C3H mice. NG-Nitro-L-arginine given to mice between 15 and 60 min prior to radiation afforded significant protection from whole body irradiation, e.g., the estimated whole body irradiation dose required to kill 50% of mice by 30 days after radiation (LD50/30) in mice treated with NG-nitro-L-arginine 60 min before irradiation was 1051 cGy compared with a whole body radiation LD50/30 of 822 cGy in control mice (P < 0.00001). Treatment of mice with DEA/NO prior to whole body irradiation also significantly reduced toxicity; the estimated whole body radiation LD50/30 was 1063 and 945 cGy in mice treated with DEA/NO 10 or 30 min before irradiation, respectively (P < 0.00001 for radiation LD50/30 of either DEA/NO-treated group compared with control). Measurement of [14C]etanidazole binding to bone marrow demonstrated that DEA/NO and NG-nitro-L-arginine exacerbated bone marrow hypoxia. Perturbations of NO levels have profound effects on in vivo radiosensitivity of normal tissues. We hypothesize that alterations in regional blood flow may underlie the changes in radiosensitivity that we have observed.

Amino Acid Oxidoreductases↗
PubMed1994

Nitrite-induced mutations in a forward mutation assay: influence of nitrite concentration and pH.

The mutagenicity of sodium nitrite at three pHs (7.4, 6.4 and 5.4) has been investigated by treating a shuttle vector plasmid in vitro and assaying for mutations within the supF target gene following replication of the damaged plasmid in human Ad293 cells. Mutation frequency increased with increasing nitrite concentration and decreasing pH. Among treatments from which a significant number of mutants could be collected, the most commonly induced mutations were GC-->AT transitions (44-56% of total mutations), followed by GC-->TA transversions (24-30%). The types of mutations induced at different nitrite concentrations and different pH's were similar, though some differences in their distribution throughout the supF gene were noted. These results provide information on the types of mutations that may be produced following the processing of nitrite-induced DNA damage in human cells.

Base Sequence↗
PubMed1994

Diethylamine/nitric oxide (NO) adduct, an NO donor, produces potent pulmonary and systemic vasodilation in intact newborn lambs.

Nitric oxide (NO), a labile humoral factor produced by vascular endothelial cells, is a potent vasodilator and an important mediator of pulmonary vascular tone. Nucleophile/NO adducts are a new class of compounds that spontaneously and predictively release NO. We investigated the hemodynamic effects of intravenous (i.v.) infusions of a recently developed NO-donor drug, the diethylamine-nitric oxide adduct (DEA/NO), in 17 intact newborn lambs. At rest, DEA/NO (1-2 microgram.kg-1.min-1) produced dose-dependent decreases in mean pulmonary (from 10.6 +/- 8.6 to 21.2 +/- 7.9%, p < 0.05) and systemic arterial pressure (from 13.2 +/- 11.7 to 31.0 +/- 15.4%, p < 0.05). Similarly, during pulmonary hypertension induced by infusion of U46619, DEA/NO (0.5-2.0 micrograms.kg-1.min-1) produced dose-dependent decreases in mean pulmonary (from 7.3 +/- 5.6 to 24.1 +/- 13.3%, p < 0.05) and systemic arterial pressure (from 2.2 +/- 3.8 to 20.3 +/- 12.9%, p < 0.05). Cardiac output (CO), heart rate (HR), systemic arterial blood gases, and pH were unchanged; atrial pressures decreased at higher doses. Equimolar infusions of S-nitroso-N-acetyl-penicillamine, nitroglycerin (NTG), and sodium nitroprusside (SNP) produced similar decreases in pulmonary and systemic arterial pressure. The nucleophile/NO adducts are potent vasodilators; their predictable and quantitative release of NO make them potentially useful research tools. In addition, because these compounds may decrease the incidence of tolerance and the risk from toxic metabolites associated with use of other nitrovasodilators, they may be clinically useful.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Constitutive expression of inducible nitric oxide synthase in human bronchial epithelial cells induces c-fos and stimulates the cGMP pathway.

Two major roles have been defined for nitric oxide (NO): cell-cell communication mediated by the stimulation of cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and cytotoxicity by direct or indirect interaction of the free radical NO with cellular targets. Thus, pathologic states might result from an alteration of NO pathways, e.g., by deregulated activity of NO synthase. To investigate this hypothesis, we introduced the murine-inducible NO synthase (iNOS) sequence into immortalized human bronchial epithelial cells (BEAS-2B). iNOS activity, measured by conversion of [14C]arginine to [14C]citrulline in the presence of 1 mM EGTA, was higher than 100 pmol/min/mg protein in early passages of iNOS-transfected cells but decreased with cell subculturing. No iNOS activity could be detected in control vector-transfected cells. NO stimulated cGMP production in iNOS-transfected cells, and this effect was inhibited by the iNOS inhibitor NG-monomethyl-L-arginine. In addition, NO production induced c-fos expression and did not interfere with clonal cell growth. These results suggest that BEAS-2B cells constitute a suitable model to study the consequences of iNOS activity on signal transduction pathways in bronchial epithelium.

Amino Acid Oxidoreductases↗
PubMed1993

Hypoxic mammalian cell radiosensitization by nitric oxide.

The bioregulatory molecule, nitric oxide (NO), was evaluated as a hypoxic cell radiosensitizer. Authentic NO gas was nearly as effective as oxygen in radiosensitizing hypoxic Chinese hamster V79 lung cells as evaluated using clonogenic assays. When NO was delivered to hypoxic Chinese hamster V79 cells using the NO-releasing agent (C2H5)2N[N(O)-NO]- Na+, radiosensitization was also observed with a sensitizer enhancement ratio of 2.4 (1 mM (C2H5)2N[N(O)NO]-Na+). Aerobic radiosensitivity was not affected at this concentration. The hypoxic cell radiosensitization properties of (C2H5)2N[N(O)NO]-Na+, coupled with the vasodilatory effects of NO on tumor vasculature, suggest that such agents open a new avenue of research in radiation oncology.

Animals↗
PubMed1993

Complexes of nitric oxide with nucleophiles as agents for the controlled biological release of nitric oxide: antiplatelet effect.

Nitric oxide (NO) inhibits platelet aggregation. Accordingly, we hypothesized that complexes of diethylamine and spermine with NO (DEA/NO and SPER/NO, respectively), two vasodilators previously shown to release NO spontaneously in aqueous solution, may also be useful antiplatelet agents. Platelet aggregation was measured in whole blood or platelet-rich plasma by impedance aggregometry after addition of collagen. In whole blood, the dose response curve for DEA/NO added 1 min before collagen was similar to that for aspirin (60% inhibition at 10(-4) M), while SPER/NO and sodium nitroprusside were less potent by an order of magnitude. In platelet-rich plasma, 10(-6) M DEA/NO caused 60% inhibition, while SPER/NO and sodium nitroprusside were as active only at 10(-5) M; aspirin's potency was unchanged from that in whole blood. In vivo, DEA/NO and sodium nitroprusside produced significant platelet inhibition 1 min after intravenous injection in the rabbit at 50 nmol/kg. Similar in vivo platelet inhibition was observed with SPER/NO and aspirin, but only at higher dose. The effects of DEA/NO and sodium nitroprusside were transient, lasting less than 30 min after treatment, while the activity of SPER/NO and aspirin was sustained throughout the 30 min experiment. The magnitude and duration of the antiplatelet effects of DEA/NO and SPER/NO correlate with the rates at which they release nitric oxide spontaneously in aqueous solution. Thus, NO/nucleophile complexes merit further exploration both as research tools and as potential antiplatelet agents.

Animals↗
PubMed1993

Nitric oxide/nucleophile complexes inhibit the in vitro proliferation of A375 melanoma cells via nitric oxide release.

Cell-mediated antitumor effects have, in part, been attributed to the production of NO. Compounds which generate NO might, therefore, be useful in attenuating the growth of tumor cells. Six nitric oxide/nucleophile adducts that release NO spontaneously in solution were tested for their effectiveness in inhibiting DNA synthesis in A375 human melanoma cells. The complexes of NO with spermine, 3-(n-propylamino)propylamine (PAPA/NO), and diethylamine reduced thymidine incorporation by 50% at concentrations of 24, 44, and 128 microM, respectively. The degree of inhibition was, in general, related to the rate and extent of NO release in solution. A melanoma cell clone sensitive to interleukin 1-mediated cytostasis (A375-C6) was no more sensitive to PAPA/NO than a clone resistant to interleukin 1 (A375-C5), suggesting that the differing inhibitory effects of interleukin 1 in the two A375 cell clones are not due to a differential sensitivity to nitric oxide. Oxymyoglobin (125 microM), a known scavenger of NO, restored the ability of A375-C6 cells to incorporate thymidine in the presence of up to 200 microM PAPA/NO. When PAPA/NO was added to a solution of oxymyoglobin, nitrosylmyoglobin was formed, indicating that the protective effect of myoglobin was due to scavenging of NO. The results are consistent with a nitric oxide-mediated mechanism for NO/nucleophile cytostasis and suggest that such compounds may be useful as tools for investigating the role of reactive nitrogen intermediates in cytostasis and cytotoxicity.

Antineoplastic Agents↗
PubMed1993

Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium.

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His+ revertants to a maximum of 4 times over background at a GTN dose of 5 mumol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 mumol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C-->T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.

Biotransformation↗
PubMed1993

Mutations induced by saturated aqueous nitric oxide in the pSP189 supF gene in human Ad293 and E. coli MBM7070 cells.

Nitric oxide is an important bioregulatory agent that may also be an endogenous and exogenous human mutagen. In order to study mutations generated following exposure of a shuttle vector-borne target gene to nitric oxide, mutations were induced in the supF gene of the pSP189 shuttle vector by treatment with nitric oxide in aerobic buffered solution followed by replication of the plasmid in either human Ad293 or Escherichia coli MBM7070 cells. The induced mutation frequency, which increased with nitric oxide dose, was 44-fold greater than the spontaneous background in human cells and > 15-fold greater than background in the bacterial cells when a total of 100 mmol of nitric oxide was oxidatively absorbed/I of pH 7.4 buffer containing the plasmid. The majority of point mutations analysed (61 and 75% for human and E. coli cells respectively) were AT-->GC transitions with GC-->AT transitions (29 and 23%) being the next most prevalent. The overall frequencies of the various point mutations seen in the supF gene were similar in the two cell types, although the distribution of hotspots showed differences. The results are consistent with a mutational mechanism initiated by deamination of DNA bases.

Base Sequence↗
PubMed1993

Mechanism of vascular relaxation induced by the nitric oxide (NO)/nucleophile complexes, a new class of NO-based vasodilators.

Compounds formed by reacting nitric oxide (NO) with various nucleophiles have been shown to dilate aortic segments with a potency that correlates strongly with the amount of NO they release spontaneously in aqueous buffers. We performed experiments aimed at confirming their mechanism of action and using the data to design improvements in their pharmacologic properties. That the vasorelaxant action these agents induce is endothelium-independent was demonstrated by exposure of denuded versus intact aortic segments to the diethylamine/NO complex (DEA/NO); denudation had no significant effect on potency. Similarly, NG-monomethyl-L-arginine, an NO synthase inhibitor, did not affect the action of DEA/NO. However, both the vasorelaxant potency of DEA/NO and the amount of cyclic guanosine monophosphate it induced were significantly diminished by the guanylate cyclase inhibitor, methylene blue. The results support the view that the NO/nucleophile adducts induce vasodilation by spontaneously releasing NO, which then activates guanylate cyclase. This mechanistic conclusion suggests that not only potency but also duration of action, a clinically relevant parameter not studied in the previous investigation, might also be controllable by structural modification. We tested this hypothesis by comparing DEA/NO and the spermine/NO adduct (SPER/NO), whose half-lives (t1/2) are 2.1 and 39 min, respectively, for persistence of their dilatory effects. The response to DEA/NO rapidly peaked (maximum at 5 min) and receded during the 60-min observation period; SPER/NO required 15 min to reach peak relaxation but maintained this level throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Complexes of nitric oxide with nucleophiles as agents for the controlled biological release of nitric oxide: hemodynamic effect in the rabbit.

Nitric oxide (NO) and drugs that generate NO are potent vasodilators. We investigated the in vivo vasodilator potential of a unique group of compounds that release NO spontaneously in solution, the NO/nucleophile complexes. The hemodynamic effects of diethylamine/NO complex (DEA/NO) (half-life < 3 min) and the spermine/NO complex (SPER/NO) (half-life > 30 min) administered intravenously were compared with those of sodium nitroprusside in anesthesized New Zealand white rabbits. Arterial pressure and systemic vascular resistance decreased, but central venous pressure, pulmonary arterial pressure, heart rate, and thermodilution cardiac output did not change in association with any of the three drugs in the doses administered. The hemodynamic effects were dose dependent. As predicted from their rates of spontaneous NO release, DEA/NO is a short-acting vasodilator with a duration similar to that of sodium nitroprusside, whereas SPER/NO is a longer-acting agent with a significant hypotensive effect 30 min after bolus injection. DEA/NO was equipotent to sodium nitroprusside, causing significant reductions in blood pressure and systemic vascular resistance at the lowest dose tested (1.5 nmol/kg). The data indicate that the NO/nucleophile complexes have predictable vasorelaxant effects in vivo as well as in vitro and suggest that their amenability to facile structure-reactivity and structure-activity modification should allow the design of NO donor drugs for a variety of applications in cardiovascular pharmacology.

Animals↗
PubMed1993

Nitric oxide/nucleophile complexes: a unique class of nitric oxide-based vasodilators.

Complexes of nitric oxide (NO) with nucleophiles, also known as nitric oxide/nucleophile adducts or NONOates, appear to offer many advantages as research tools in cardiovascular pharmacology and may have future clinical potential as well. A wide variety of NONOates can be synthesized simply by exposing various nucleophilic compounds to NO. The products are generally stable as solids and highly soluble in aqueous media. The potent vasodilator activity displayed by select members of this series is endothelium independent and is mediated by the free NO that is released on dissolution, which activates smooth-muscle guanylate cyclase with subsequent intracellular cyclic guanosine monophosphate production. NO release from the NONOate complexes is not catalyzed by exogenous thiol or albumin. The NONOates differ from other currently available nitrovasodilators in that their potency as vasorelaxants correlates closely with data on their first-order rates of spontaneous reversion to NO in simple aqueous buffers. The compounds' properties can be conveniently altered by changing the identity of the nucleophilic residue. Continued work with NONOate complexes may provide useful clinical agents as well as improved tools for probing the bioregulatory roles of NO.

Animals↗
PubMed1991

Acceleration of N-nitrosation reactions by electrophiles.

Selected mechanisms by which electrophiles can facilitate N-nitrosamine formation are reviewed. Special attention is given to a recently discovered reaction in which nitrogen in its lowest (ammonia) oxidation state is efficiently converted to N-nitroso compounds by oxidation in the presence of secondary amines; an electrophilic transition metal centre (E+) makes this reaction possible by initially N-coordinating the ammonia (E+ + NH3----E-NH3+). Other mechanisms considered include: the conversion of nitrite under nonacidic conditions via nitro complexes to nitrosatively active transition metal nitrosyl intermediates (E+ + NO(2-)----E-NO(2-)----E-NO2+); catalysis of N-nitrosamine formation in nitrite-amine mixtures by electrophilic carbon centres that initiate the reaction by attack on the amine (E+ + R2NH----E-NR2); and coordination of nitrite by carbon electrophiles to form activated O-bound species (E+ + ONO(-)----E-O-NO) capable of performing the required N-nitrosation. The findings suggest that acceleration of N-nitrosamine-forming reactions by electrophiles may be a critical factor to consider in attempting to rationalize, predict and control the distribution of carcinogenic N-nitroso compounds in vivo and in the environment.

Chemical Phenomena↗
PubMed1991

Carcinogenicity study of fecapentaene-12 diacetate on skin painting in SENCAR mice.

To investigate the effects of both diol esterification and coadministration with antioxidant on the tumorigenicity of fecapentaene-12 (FP-12) preparations, diacetylfecapentaene-12 (DAFP-12) in dimethylsulfoxide (DMSO) was applied to SENCAR mouse skin with or without the stabilizer, vitamin E, twice/week for 5 weeks, following which all animals were promoted for up to 25 weeks by weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). While positive controls receiving 7,12-dimethylbenz[a]anthracene (DMBA) instead of DAFP-12 in a similar protocol all developed papillomas (average of 23/animal), papilloma incidence in mice given DAFP-12 did not differ significantly from that of the vehicle control. We conclude that DAFP-12 shows little or no tumor initiating activity for mouse skin even when coadministered with vitamin E.

Adult↗