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Biomedical subjects

R D Snyder

Publications and source records attributed to R D Snyder.

At least 73 records · Page 4Linked to original sources
PubMed1987

Effects of sodium selenite on DNA and carcinogen-induced DNA repair in human diploid fibroblasts.

Sodium selenite induces substantial DNA damage in human fibroblasts. This damage appears to consist of true DNA breaks rather than alkali-labile sites and to not arise via free oxygen radical production. Cloning efficiency and DNA strand breakage show dramatically enhanced sensitivity to selenite if the treatments are carried out in the presence of reduced glutathione or, to a lesser degree, serum, supporting the notion that a glutathione-selenite conjugant is required for activation to a genotoxic form. In addition, the notion that selenium anticarcinogenicity involves enhancement of cellular DNA repair, has been examined. No evidence for enhancement (or inhibition) of repair of methyl methanesulfonate (MMS)-, UV- or bleomycin-induced DNA damage was observed in human fibroblasts treated with selenite.

Bleomycin↗
PubMed1986

Neurofibromatosis xenografts. Contribution to pathogenesis.

We transplanted Schwann cells of 3 patients with neurofibromatosis from neurofibromas, sural nerve, and from a malignant schwannoma into sciatic nerves of immunoincompetent mice. Three and six months later, the grafts and distal nerve segments contained normal myelinated fibers. After rendering host animals immune competent again, neurofibroma and malignant schwannoma Schwann cells were rejected, but grafts retained normally myelinated fibers indicating that these were of mouse origin. Sural nerve Schwann cells from a neurofibromatosis patient were rejected also leaving naked axons in the grafted segments showing that human Schwann cells from the sural nerve of one patient had invested and myelinated the regenerating mouse axons. The nature of putative signals passing between axons and Schwann cells might be elucidated by the combination of human and animal cells in immunoincompetent host nerves. Hypothetical signals for myelination of mouse axons were normally received by sural nerve Schwann cells of a patient with neurofibromatosis, but not by Schwann cells from neurofibromas or malignant schwannomas.

Animals↗
PubMed1986

Evaluation of putative inhibitors of DNA excision repair in cultured human cells by the rapid nick translation assay.

A human fibroblast nick translation assay has been applied to an examination of 48 diverse chemical agents to assess their ability to specifically interfere with the DNA excision-repair process following ultraviolet irradiation. Certain inhibitors of DNA polymerase, ribonucleotide reductase and purine and pyrimidine biosynthesis are shown to inhibit the resynthesis step of repair while DNA intercalators and inhibitors of DNA topoisomerases appear to inhibit the incision step. A variety of other agents previously implicated as inhibitors of DNA repair was also examined and found to have no such effect. This type of analysis should prove useful in the rapid identification of new classes of compounds that antagonize normal cellular repair functions and that might, therefore, act as comutagens or cocarcinogens.

Cells, Cultured↗
PubMed1986

Genotoxicity of formaldehyde and an evaluation of its effects on the DNA repair process in human diploid fibroblasts.

Formaldehyde treatment of human fibroblasts gave rise to DNA damage detected by a nick translation assay. This damage was not repaired by typical 'long-patch'-type excision repair as evidenced by the failure of DNA repair inhibitor post-treatment to elevate the amount of DNA strand breakage. In addition, the effects of formaldehyde on DNA repair were examined in light of a recent report suggesting that formaldehyde inhibited the repair of X-ray-induced strand breaks and UV- and benzo [a]pyrene diol epoxide-induced unscheduled DNA synthesis in human bronchial cells. We report that formaldehyde (1) was ineffective at inhibiting the sealing of X-ray- or bleomycin-induced DNA strand breaks, (2) did not inhibit the removal of pyrimidine dimers from cellular DNA at short treatment times, and (3) that the previously observed inhibition of unscheduled DNA synthesis was most likely due to the inhibition of uptake of labeled precursor into formaldehyde-treated cells. Thus, our findings are not consistent with the notion that formaldehyde inhibits the repair process in human fibroblasts. Finally, formaldehyde was shown to elevate the level of misincorporation of bases into synthetic polynucleotides catalyzed by E. coli DNA polymerase I, indicating that the mutagenicity of formaldehyde may be due to covalent alteration of DNA bases.

Bleomycin↗
PubMed1985

Nick translation--a new assay for monitoring DNA damage and repair in cultured human fibroblasts.

An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. This nick translation assay, originally proposed by Nose and Okamoto [1983], is very sensitive, allows detection and quantitation of both DNA damage and repair, distinguishes between various types of induced damage, does not require radioactive prelabeling of cells, and circumvents some of the problems inherent in unscheduled DNA synthesis (UDS) assays. The assay is also useful in detecting those agents that inhibit replicative DNA synthesis and/or the excision repair process. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

Carcinogens↗
PubMed1985

Effects of nucleotide pool imbalances on the excision repair of ultraviolet-induced damage in the DNA of human diploid fibroblasts.

Recent studies in our laboratory and others have demonstrated that DNA polymerase inhibitors such as the ara nucleosides, aphicolin and dideoxythymidine are potent inhibitors of the DNA excision repair process in confluent human fibroblasts as evidenced by the agent-dependent accumulation of single-strand interruptions in the DNA of UV-irradiated, but not in unirradiated, cellular DNA. In rapidly cycling cells, on the other hand, these agents are weak inhibitors at best but when used in combination with the ribonucleotide reductase inhibitor, hydroxyurea, a significant enhancement of inhibitory capacity is seen. In an attempt to better understand the mechanism of repair inhibition by DNA polymerase inhibitors, and the nature of this hydroxyurea enhancement, experiments were initiated in which the effects of a series of ribonucleotide reductase inhibitors on dNTP pools and on the DNA repair process were determined in both quiescent cultures and log-phase cultures of human fibroblasts. It was determined that hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone (TSC), pyrozoloimidazole (IMPY), 3,5-diamino-1,2,4-triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid (THBA) and 3,4-dihydroxy benzohydroxamic acid (DHBA) are all effective inhibitors of the DNA repair process in confluent cells but not in log-phase cells. Moreover, the effects of these inhibitors can be reversed by the addition of certain combinations of deoxynucleosides. These reversal studies and the direct analysis of dNTP pool modulation by these compounds in log phase and confluent cultures support the notion that specific pool depletions rather than general imbalance of pools gives rise to the inhibition of the DNA excision repair process.

Cells, Cultured↗
PubMed1985

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine.

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m2). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10(8) daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2-9.4 breaks/10(8) daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2-14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10(8) daltons, respectively). The same level of repair inhibition was observed in confluent XP-variants receiving ara-C/HU, but was reduced by 62-68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.

Cells, Cultured↗
PubMed1985

An examination of the DNA damaging and repair inhibitory capacity of phorbol myristate acetate in human diploid fibroblasts.

The effects of the tumor promoter, phorbol myristate acetate (TPA) on DNA and DNA repair in human diploid fibroblasts was investigated. TPA induced detectable DNA single-strand breaks at doses as low as 1.3 microM using a sensitive nick-translation assay and at doses as low as 40 microM using alkaline sucrose velocity sedimentation analysis. This report provides the first indication of TPA-induced DNA damage in any cell other than a leucocyte. Experiments utilizing enhancers or inhibitors of active oxygen generation suggest that the DNA damage is related to free radical formation. In addition, we have investigated the effects of TPA on survival following u.v.-irradiation and on removal of pyrimidine dimers from cellular DNA and find no indication of enhancement of u.v. sensitivity or inhibition of normal DNA repair processes in these cells. In fact, TPA treatment enhances colony-forming ability of u.v.-irradiated, but not unirradiated cells.

Cells, Cultured↗
PubMed1985

Intranasal cocaine abuse in an allergists office.

Fourteen adults complaining of persistent nasal congestion were found to be recreational cocaine users. All eventually admitted to intranasal drug snorting. None responded to routine nasal therapy.

Administration, Intranasal↗
PubMed1985

Effects of hydroxyurea and thymidine derivatives on the uptake and metabolism of deoxycytidine and arabinofuranosylcytosine in log phase and contact-inhibited human diploid fibroblasts.

Hydroxyurea and pyrimidine analogs have been shown to enhance the chemotherapeutic efficacy and the DNA excision repair-inhibitory capacity of arabinofuranosylcytosine (ara-C). Since various cell types are expected to respond differently to these combination treatments and since little is known about the nature of their antiproliferative effects, we have investigated the metabolism and uptake into acid-soluble pools of deoxycytidine (dCyd) and ara-C in cycling and non-cycling human diploid fibroblasts. A substantial fraction of dCyd is converted through deamination to deoxyuridine and thymidine nucleotides, and this occurs to a greater degree in log phase cultures. ara-C is more resistant to deamination and is metabolized primarily to ara-CTP. Hydroxyurea decreases the proportion of dCyd and ara-C which is deaminated under both growth conditions, leading to higher levels of ara-CTP and dCTP. Trifluorothymidine causes an accumulation of dUMP and decreases the formation of dCTP in log phase and confluent cells. Thymidine inhibits deamination in log phase cells but stimulates this pathway in noncycling cells. Dideoxythymidine did not appreciably alter the spectrum of metabolites of dCyd formed in log or confluent phase cells but was shown to inhibit the transport of dCyd and thymidine across the membrane. These studies provide information regarding the nature of the enhancement of the antiproliferative activity of ara-C by commonly used drugs and indicate that the cycling state of the target cell plays a major role in determining the metabolism of the nucleoside and the efficacy of chemotherapeutic treatments.

Arabinofuranosylcytosine Triphosphate↗
PubMed1984

3-Aminobenzamide does not alter DNA repair in human fibroblasts through modulation of deoxynucleoside triphosphate pools.

3-Aminobenzamide does not deplete cellular purine deoxynucleoside triphosphate pools as do the structurally-related ribonucleotide reductase inhibitors, the hydroxy- and amino-substituted benzohydroxamic acids. Thus, the previously reported ability of 3-aminobenzamide to inhibit de novo synthesis of DNA purines does not appear to be due to a direct effect on pools via inhibition of ribonucleotide reductase. The enhancement rather than inhibition by 3-aminobenzamide of DNA repair in the present studies, however, leaves open the possibility that pool modulation may play a role in cell systems where repair inhibitory effects are seen.

Antineoplastic Agents↗
PubMed1984

Deoxyribonucleoside triphosphate pools in human diploid fibroblasts and their modulation by hydroxyurea and deoxynucleosides.

Deoxyribonucleoside triphosphate (dNTP) pool levels were examined in synchronized and unsynchronized log phase cultures and in quiescent cultures of human diploid foreskin fibroblasts. dNTP levels were in good agreement with those previously published for human HeLa and lymphoblastic leukemia cells. dCTP and dGTP levels showed only a modest lowering in quiescent as compared to log-phase cells, but dATP and dTTP levels were reduced dramatically in quiescent cultures. Cells synchronized by serum starvation and assayed at the peak DNA synthetic phase (18-21 hr post release) showed substantially higher pools of all four dNTPs. Hydroxyurea treatment reduced only purine dNTPs in both log phase and confluent cells while increasing dTTP and dCTP pools. The effects of deoxynucleosides on dNTP pools were also examined and are discussed in light of current models regarding regulation of purified ribonucleotide reductase formulated from in vitro studies.

Cells, Cultured↗
PubMed1984

Inhibitors of ribonucleotide reductase alter DNA repair in human fibroblasts through specific depletion of purine deoxynucleotide triphosphates.

Hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone, pyrazoloimidazole, 3,5-diamino-1,2,4 triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid and 3,4-dihydroxy benzohydroxamic acid were examined for their effects on cellular dNTP pools, DNA excision repair, DNA replication and deoxynucleoside uptake in human diploid fibroblasts. All 7 agents were effective inhibitors of the UV excision repair process in noncycling quiescent cells, but not in rapidly dividing log-phase cells. This differential effect clearly demonstrates dependency upon modulation of cellular purine dNTP pool levels at the level of the reductase. Repair synthesis is shown to be less sensitive to all 7 reductase inhibitors than is replicative synthesis. Studies on cellular uptake of labeled DNA precursors in inhibitor-treated cells support the notion that deoxynucleosides cannot channel into the replicative synthesis process whereas they are readily utilized at repairing sites.

Cells, Cultured↗
PubMed1984

The inhibition of ultraviolet radiation-induced DNA repair in human diploid fibroblasts by arabinofuranosyl nucleosides.

The antiviral compounds 9-beta-D-arabinofuranosyladenine (ara-A), 9-beta-D-arabinofuranosyl-2-fluoroadenine (FAA), 9-beta-D-arabinofuranosylhypoxanthine (ara-Hx), 9-beta-D-arabinofuranosylguanine (ara-G), 1-beta-D-arabinofuranosylthymine (ara-T), 1-beta-D-arabinofuranosyl-2'-fluorocytosine (FAC), 1-beta-D-arabinofuranosyl-2'-fluoro-5-iodocytosine (FIAC) and 1-beta-D-arabinofuranosyl-2'-fluoro-5-methyluracil (FMAU) were compared to 1-beta-D-arabinofuranosyl cytosine (ara-C) in their ability to inhibit ultraviolet (UV) light-induced DNA repair in log phase and confluent human diploid fibroblasts. Inhibition of the polymerization or ligation steps of DNA excision repair manifests itself in the form of DNA single-strand breaks which may be quantitated through velocity sedimentation analysis in alkaline sucrose gradients. In UV-irradiated quiescent, confluent human fibroblast cultures, treatment with any of the aranucleosides leads to accumulation of single-strand breaks but the effective dose for this inhibition varies greatly. The order of their effectiveness in confluent cultures was ara-C and its derivatives greater than ara-A, FAA, ara-G, ara-Hx greater than ara-T. In rapidly cycling cells on the other hand, sensitivity to repair inhibition was exhibited only in response to ara-C and FAC. If 2 mM hydroxyurea (HU) was administered with ara-A, FAA or FMAU, however, DNA strand breaks were seen. HU also increased the efficiencies of ara-C and FAC. No significant strand breaks were observed in UV-irradiated log phase cells treated with FIAC, ara-Hx, ara-G or ara-T even in the presence of HU. The efficiencies of inhibition of unscheduled DNA synthesis (UDS) and semiconservative DNA synthesis by the aranucleosides is consistent with their relative efficiencies at producing strand breaks. The ability of the aranucleosides to inhibit DNA repair is discussed with respect to their hypothesized effects on DNA metabolic processes in eucaryotic cells.

Arabinonucleosides↗
PubMed1984

Ventriculomegaly in childhood bacterial meningitis.

Sixty-seven of 125 children with bacterial meningitis had computerized tomography (CT) of the head. Ventriculomegaly was the most common abnormality on CT occurring in twenty-eight cases. Progressive enlargement of the ventricular system occurred without evidence of persistent increased intracranial pressure, although early in the illness increased intracranial pressure was usually present. Factors other than persistent increased pressure appear to cause ventriculomegaly in some cases. Ventricular enlargement on CT in the absence of clinical evidence of increased intracranial pressure may not be an indication for surgical intervention.

Cerebral Ventricles↗