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Biomedical subjects

H G Claycamp

Publications and source records attributed to H G Claycamp.

29 records · Page 2Linked to original sources
PubMed1990

Superoxide dismutase and media dependence of far-UV radiation resistance in thiol-treated cells.

Pretreatment of wild-type Escherichia coli K12 cells with dithiothreitol (DTT) induces far-UV radiation resistance after the thiol is removed (Claycamp 1988). The present study shows that a 1 h treatment of cells with DTT in minimal medium followed by a 0.5 h incubation in buffer (37 degrees C) results in a dose reduction factor (DRF) calculated at F37 of 1.81. When the thiol pretreatment was in rich medium, sensitization occurs with DRF = 0.729. This sensitization could be reversed to protection by inhibiting extracellular thiol oxidation in rich medium with the chelator, DETAPAC, such that the thiol oxidation rate was equivalent to that of DTT in minimal medium. Both thiol-induced resistance and sensitization produced changes predominantly in the shoulders of the survival curves. Furthermore, for either protection or sensitization, at least one form of endogenous superoxide dismutase (SOD) was required: in SOD-deficient cells (sodAsodB) the DRFs were 1.08 and 0.882 for minimal and rich media, respectively. These results suggest that different targets are involved in thiol-induced UV protection and sensitization: DNA and extracellular targets (e.g. the membrane), respectively. The results augment observations of alternate and multiple repair pathways inducible by oxygen radicals and may help understanding non-physicochemical thiol protection mechanisms.

Cell Survival↗
PubMed1990

The dependence of thiol-inducible radiation resistance in Escherichia coli K12 on the medium and catalytic metal.

Radiation protection by thiols in procaryotes and lower eucaryotes has been demonstrated repeatedly to require a competent DNA repair phenotype, suggesting that simple chemical radical scavenging and hydrogen donation are only a portion of the mechanism of radiation protection by thiols. In the present report, thiol-induced radiation resistance--a model in which cells are pretreated with dithiothreitol and then irradiated in the absence of thiol--is shown to be a medium-dependent process. Wild-type log-phase cells treated with dithiothreitol in minimal-glucose medium are induced to radioresistance that persists after the thiol has been removed. Although the thiol pretreatment affected the antioxidants (catalase, superoxide dismutase, and glutathione) in cells at the time of irradiation, various antioxidant levels did not predict radiation resistance. Thiol-induced radioresistance is not expressed in rich medium-treated cells or in DNA repair (recA)-deficient cells. Addition of the efficient chelator, DETAPAC, to the thiol treatment medium leads to additional radioresistance in the case of minimal medium and a moderate expression of resistance in rich medium. Experiments using the intracellular chelator, 1,10-phenanthroline, in the presence of thiol led to inhibition of thiol-induced resistance in minimal medium and radiosensitization in rich medium. These results can be explained by a "site-specific" mechanism of thiol oxidation in which the chelators control the site(s) and rate of thiol oxidation, subsequently determining the type of cellular response.

Culture Media↗
PubMed1989

Dimethylformamide-induced changes in the radiation survival of low- and high-passage intestinal epithelial cells (IEC-17) in vitro.

The effects of dimethylformamide (DMF) on the radiation response of low- and high-passage intestinal epithelial cells (IEC-17) were examined. The IEC-17 cell line, a rat intestinal epithelial cell line, exhibited a bimodal response to X radiation. The sensitive fraction, which was attributed to a stem cell-like component, had a D0 of 0.90 Gy. The resistant fraction, thought to be the expression of a more mature component, exhibited a D0 of 2.00 Gy. Treatment using a putative cell differentiating agent, N,N-dimethylformamide, increased the resistant fraction of the population from 35 to 80%, suggesting that DMF treatment (100 mM) increased the proportion of mature cells in the IEC-17 cell population. In addition, extended age in culture (greater than 100 passages) resulted in altered morphology, decreased doubling time, increased chromosome number, and loss of anchorage dependence, all features characterizing spontaneously transformed high-passage IEC-17 cells. These high-passage cells also exhibited a bimodal response to X radiation; the sensitive fraction had a D0 of 0.80 Gy while the resistant fraction D0 was 1.50 Gy. DMF increased the resistant fraction from 35 to 55% of the population. Results suggested that the different radiosensitivities of the subpopulations remained throughout the spontaneous transformation of high-passage IEC-17 cells.

Animals↗
PubMed1988

Enhanced survival of gamma-irradiated Escherichia coli following pretreatment with dithiothreitol.

Survival of three strains of Escherichia coli K12 was studied with respect to radiation protection by dithiothreitol (DTT). The three strains compared were AB2462 recA, AB2470 rec21 and their DNA repair-competent prototype, AB1157. The strains were incubated in 10 mmol dm-3 DTT for 60 min and allowed an expression period for SOS functions to appear which may have been induced by DTT. Following the expression period the DTT-incubated cells and incubated control cells were irradiated. When AB1157 cells were pretreated with chloramphenicol (200 micrograms cm-3) for a period of 30 min prior to addition of the induction media no increase in survival was seen. When catalase (0.1 mg cm-3) was added to the AB1157 cells prior to the induction media a decrease in the degree of induction was noted with an enhancement ratio (ER) of 0.893 (ER-1 = 1.12). Furthermore, DTT-treated AB2462 and AB2470 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with or without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a dose modification factor (DMF) of 1.7 with DTT present and 1.3 with pretreatment; (2) the rec mutants showed no change in survival at any dose with a DMF of approximately 1.0. Results indicate that, using our protocol, inducible repair is of more importance than free radical scavenging by DTT. Furthermore, DTT-treated AB2462 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with and without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a DMF of 1.7 with DTT present during irradiation and 1.3 with only pretreatment; (2) the recA and recB mutants showed no change in cell survival at any dose with a DMF of approximately 1.0. Results indicate that, using our pretreatment protocol, inducible repair is of more importance in protection than free radical scavenging by DTT.

Catalase↗
PubMed1988

Dithiothreitol pretreatment and inducible repair in UV-irradiated Escherichia coli K12 cells.

The UV radiation survival of several Escherichia coli K12 strains was measured after pretreatment of the cells with dithiothreitol (DTT). In DNA repair-competent cells (AB1157), UV survival was enhanced (ER = 1.2) after pretreating cells for 1.0 h using 10 mmol dm-3 DTT and then incubating the cells for 1.5 h in buffer before UV irradiation. Similar experiments using the excision repair mutant, AB1886uvrA6, or the recombination repair and SOS-deficient mutant, AB2462recA, strains did not show enhanced UV survival. None of the E. coli strains tested were protected against UV killing by simultaneous treatment with DTT (10 mmol dm-3). These results, and the fact that incubation in chloramphenicol removed the wild-type response in DTT-pretreated, UV-irradiated cells, suggest that the observed UV radioprotection was a result of inducible enzymatic repair processes such as recA-dependent repair. The proposed stimulus for inducible repair in these cells is DNA damage caused by intracellular hydroxyl radicals arising from thiol oxidation. The involvement of oxygen radicals in the induction pathway is supported by results that showed superoxide dismutase and catalase could inhibit a portion (one-third) of the inducible repair.

Cell Survival↗
PubMed1988

Absence of pyrimidine salvage and prevention of thymineless radiosensitization in Escherichia coli thyA cells fed dihydrothymine or thymine glycol.

Little information is available concerning the metabolic fate of radiation-induced thymine base damage products once they have been excised from DNA. The present study was an attempt to determine whether or not thymine-requiring mutants of Escherichia coli could grow on dihydrothymine (DHT) and thymine glycol (TG) by "salvaging" the altered thymines. A second test of thymine product utilization was prevention of thymineless radiosensitization. Results showed that very low growth of Thy- cells on DHT or TG could be explained by the presence of less than or equal to 1% contaminating thymine in the mixtures. Radiation dose-modification factors (DMFs) for thyA cells fed DHT or TG for 3 h were 1.38 +/- 0.28 and 1.26 +/- 0.24, respectively, whereas the DMF for 3 h thymine-starved cells was 1.63 +/- 0.05. The small (approximately 25%) amelioration of thymineless radiosensitization observed in DHT- or TG-fed cells could probably be explained by contaminating thymine in the medium. Although DHT is a normal metabolite in some cells, neither DHT nor TG could be used efficiently by thymine-requiring cells in the protocol presented.

Dose-Response Relationship, Radiation↗
PubMed1987

Dithiothreitol-induced oxidative damage to thymine and DNA in solution.

Studies on dithiothreitol-induced oxidative damage to thymine and DNA in solution are reported. The major thymine products, cis- and trans-5,6-dihydroxy-5,6-dihydrothymine (thymine glycols), are produced rapidly in 37 degrees C neutral solutions of 10mM thymine and 10mM dithiothreitol. Iron-EDTA enhances while the iron chelator, diethylenetriaminepentaacetic acid, inhibits the reaction. In experiments using 3H-TdR-labeled Escherichia coli DNA, DNA damage was measured as increased ethanol-soluble radioactivity after treatment of the DNA with 5mM dithiothreitol at 45 degrees C. The findings are important with respect to current research interest in thiol radioprotection and thiol-plus-heat toxicity.

DNA Damage↗
PubMed1986

PDN-1: a spin-labeled analog of cis-diamminedichloroplatinum(II). Biophysical studies in aqueous, lipophilic, and biological media.

Biophysical and biochemical studies on a biradical-labeled analog of Cisplatin, PDN-1, in aqueous, biological, and lipophilic media are presented. The ESR spectra from PDN-1 in aqueous meida show five-line spectra typical of biradicals in which intramolecular spin exchange occurs. The spectrum of PDN-1 in 1-octanol is characterized by three broad lines overlying a fourth line that is approximately three times as wide as the others. Although the characteristics of the aqueous spectra differ substantially from those of the octanol spectra, both the midfield peak height and the integral of the absorption spectrum can be used as a linear measure of PDN-1 concentration in either media. The smallest concentration used in these linear calibrations was equivalent to the detection of 60 femptomoles of PDN-1 at a signal-to-noise ratio of 30. PDN-1 is shown to "decay" in tissue culture medium which involves either the reduction of a nitroxyl moiety, the labilization of an amino bond, or a combination of both. Spectra of intracellular PDN-1 show that both a mobile ("free") fraction and slowly tumbling ("bound") fraction of the compound can be detected within CHO cells. The spin-labeled cisplatin method is at least as sensitive as conventional chromatographic and spectroscopic methods, yet has the added advantage of offering information about the molecular environment of the complex.

Animals↗
PubMed1986

Spin-label substitutions in cisplatin reduce toxicity and interaction with radiation.

A piperidinyl-labeled platinum(II) complex, cis-Pt[2,2,6,6-tetramethyl-4-aminopiperidine-N-oxyl]2dichloride , is an ESR-traceable analog of the antitumor agent, cis-diamminedichloroplatinum(II), DDP. The toxicity of the analog (PDN-1) in Chinese hamster ovary (CHO) cells is 22 times less than that of the parent compound: D0 (where D0 = slope-1 of the straight-line portion of the survival curve) = 0.11 mM versus 0.0053 mM for 1.5-h treatments with PDN-1 and DDP, respectively. At PDN-1 doses that cause 10% killing when used alone, no statistically significant increase in cell killing of hypoxic, irradiated cells was observed. PDN-1 does not have significant dose-modifying properties but may be a useful probe to study platinum in mammalian cells.

Animals↗