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R M Tyrrell

Publications and source records attributed to R M Tyrrell.

At least 73 records · Page 4Linked to original sources
PubMed1990

New trends in photobiology. The interaction of UVA radiation with cultured cells.

Recent work concerning the interaction of UVA radiation (320-380 nm) with cultured cells is reviewed with particular emphasis on the involvement of cellular oxidative stress in the biological effects of this radiation on eucaryotic cells. Possible chromophores are considered and their role in generation of various oxidant species including hydrogen peroxide, superoxide anion, singlet oxygen and hydroxyl radical. DNA and membranes are discussed as possible targets for the lethal action of long wavelength radiation. Four mechanisms of cellular defence are proposed: (1) DNA repair; (2) antioxidant enzymes; (3) endogenous free radical quenchers; (4) inducible protection.

Animals↗
PubMed1990

Mutagenesis by hydrogen peroxide treatment of mammalian cells: a molecular analysis.

Hydrogen peroxide is an oxidizing agent which can be generated intracellularly either during normal metabolism or by treatment with external agents including solar UV radiation. Simian cells (CV-1) transfected with the SV40-based shuttle vector plasmid pZ189 have been treated with H2O2 and then incubated to allow repair and replication of the plasmid. The frequency of mutations at the supF locus of the recovered plasmid increases by a factor of up to four over the spontaneous value. The nucleotide changes associated with 100 spontaneous and 100 H2O2-induced mutants have been determined directly by sequencing a 150 bp fragment that includes the entire supF tRNA coding region. Deletions were observed in approximately 45% of both the spontaneous and induced mutants, whereas single or multiple base changes arose in 68 and 57% of the induced and spontaneous mutants respectively. The spectrum of induced mutations is characterized by (i) the occurrence of deletions associated with base changes (16% of all mutants analysed) and (ii) small deletions of 3 bp and less (51% of all deletion mutants sequenced). Sixty-five per cent (15 out of 23) of all small deletions (spontaneous and induced) are associated with runs of between two and five identical bases and eight of them arise at a mutational 'hotspot' region of five cytosines between bp 172 and 176. The majority (19 out of 30) of completely sequenced deletions observed in the spontaneous spectrum contain either (i) small (2-10 bp) direct repeat sequences that lie immediately outside one deletion terminus and immediately inside the second deletion terminus or (ii) small (2-3 bp) inverted repeat sequences lying immediately inside the two deletion termini. Most deletions that we have observed are therefore likely to arise as a consequence of specific aspects of DNA structure.

Animals↗
PubMed1990

Induction of the heme oxygenase gene in human skin fibroblasts by hydrogen peroxide and UVA (365 nm) radiation: evidence for the involvement of the hydroxyl radical.

The induction of heme oxygenase by both hydrogen peroxide and UVA (365 nm) radiation in normal human skin fibroblasts is prevented by prior treatment of cells with the specific iron chelators, o-phenanthroline or desferrioxamine. In addition, both iron chelators protected cells against the lethal effects of H2O2 treatment or UVA irradiation. We propose that the generation of the highly reactive hydroxyl radical by an iron catalyzed Fenton reaction is involved both in the induction of this stress response and, at least in part, in cell killing by the two treatments. These results are also consistent with the idea that the heme oxygenase gene is induced in response to oxidative stress and that its induction may constitute an inducible protective mechanism against oxidative damage induced by both hydrogen peroxide and UVA radiation.

Cell Survival↗
PubMed1990

Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts.

Treatment of cultured human skin fibroblasts with near-UV radiation, hydrogen peroxide, and sodium arsenite induces accumulation of heme oxygenase mRNA and protein. In this study, these treatments led to a dramatic increase in the rate of RNA transcription from the heme oxygenase gene but had no effect on mRNA stability. Transcriptional activation, therefore, appears to be the major mechanism of stimulation of expression of this gene by either oxidative stress or sulfydryl reagents.

Actins↗
PubMed1989

The spectrum of mutations generated by passage of a hydrogen peroxide damaged shuttle vector plasmid through a mammalian host.

Treatment of a plasmid shuttle vector (pZ189) with a combination of hydrogen peroxide and a ferric iron/EDTA complex prior to transfection and passage in simian (CV-1) cells increases the frequency of mutations at the supF locus by up to 60-fold over the spontaneous background. This increase in mutation frequency is abolished when the inhibitors desferrioxamine, superoxide dismutase, catalase or dimethyl sulfoxide are included in the initial reaction or when the iron/EDTA complex is omitted, a strong indication that the premutagenic damage arises as a result of direct attack by hydroxyl radical generated in a superoxide driven Fenton reaction. DNA sequence analysis of the mutated plasmids shows that 1) Deletions occuring in combination with base-substitutions arise in 22.5 percent of the induced mutants compared with only 3 percent of spontaneous mutants 2) Sixty percent of all induced deletion mutations involve the loss of a single base and 77 percent of these (20 out of 26) occur at two adenine-containing sites 3) The base-change spectrum of mutants arising in the treated plasmid population is marked by the predominance of mutants containing a single base-change and by an increase in changes at AT base pairs. These results provide direct information concerning the nature of mutations arising in mammalian cells as a result of hydroxyl radical mediated DNA damage.

Animals↗
PubMed1989

Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite.

We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage.

Amino Acid Sequence↗
PubMed1989

Singlet oxygen involvement in the inactivation of cultured human fibroblasts by UVA (334 nm, 365 nm) and near-visible (405 nm) radiations.

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.

Cells, Cultured↗
PubMed1988

Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector.

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.

Animals↗
PubMed1987

Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts.

We have analyzed the pattern of protein synthesis in solar near ultraviolet (334 nm, 365 nm) and near visible (405 nm) irradiated normal human skin fibroblasts. Two hours after irradiation we find that one major stress protein of approximately 32 kDa is induced in irradiated cells. This protein is not induced by ultraviolet radiation at wavelengths shorter than 334 nm and is not inducible by heat shock treatment of these cells. Although sodium arsenite, diamide, and menadione all induced a 32-kDa protein, they also induced the major heat shock proteins. In contrast, the oxidizing agent, hydrogen peroxide, induced the low molecular weight stress protein without causing induction of the major heat shock proteins. A comparison of the 32-kDa proteins induced by sodium arsenite, H2O2, and solar near ultraviolet radiation using chemical peptide mapping shows that they are closely related. These results imply that the pathways for induction of the heat shock response and the 32-kDa protein are not identical and suggest that, at least in the case of radiation and treatment with H2O2, the 32-kDa protein might be induced in response to cellular oxidative stress. This conclusion is supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32-kDa stress protein.

Arsenic↗
PubMed1987

Evidence for two independent pathways of biologically effective excision repair from its rate and extent in cells cultured from sun-sensitive humans.

Repair-proficient human cells can be sensitized to exposure to UV radiation at 254 nm by postirradiation incubation in the presence of the eukaryotic alpha polymerase inhibitor, aphidicolin. The degree of sensitization has been examined in cells cultured from humans suffering from various types of sun-sensitive syndromes. Xeroderma pigmentosum (XP) variant and Bloom's cell lines (both excision proficient) were strongly sensitized by aphidicolin. An excision repair proficient Cockayne's cell line and a deficient XPD line were both sensitized to a level similar to the sensitivity of excision deficient XPA cells. In contrast, three XPC cell lines which show intermediate UV-induced repair replication and UV sensitivity were sensitized little (in one case) or not at all (in two cases) to UV by postirradiation inhibition of the alpha polymerase. These results lead us to conclude that there are two independent pathways of biologically effective excision repair, the major one of which involves the alpha polymerase and a second, less efficient and slower pathway which is independent of the alpha polymerase and which is the only pathway operating in two of the three XPC strains tested. The rates of biologically effective excision repair were similar in normal, XP variant, and Cockayne's cell lines, but these rates were considerably higher than published rates of dimer excision measured under similar conditions.

Aphidicolin↗
PubMed1987

Action spectra for human skin cells: estimates of the relative cytotoxicity of the middle ultraviolet, near ultraviolet, and violet regions of sunlight on epidermal keratinocytes.

Action spectra for the cytotoxic action of electromagnetic radiation in the solar range 280-434 nm have been determined for human fibroblasts and epidermal keratinocytes derived from the same foreskin biopsy. The spectra for the two cell types are close to identical and coincide with our previously published data for a human lymphoblastoid line indicating that the mechanism of inactivation of the three human cell types is similar at any given wavelength. Using published data for ultraviolet transmission of human skin and sample spectral irradiance data, we have estimated the relative biological effectiveness of the middle ultraviolet (UVB) (290-320 nm), near ultraviolet (UVA) (320-380 nm), and violet (380-434 nm) regions of sunlight for cytotoxicity at the basal layer of the epidermis. We conclude that the UVB component in noon summer sunlight (the most UVB rich spectral conditions tested) may contribute only about 40% of the total cytotoxic effectiveness of sunlight at 290-434 nm. At lower zenith angles, UVA can account for up to 80% of the cytotoxic effectiveness of the combined UVA and UVB regions. Finally, a comparison of published action spectra data for human erythema with cytotoxicity data corrected for ultraviolet transmission to different depths of the human epidermis suggests that UVA erythema could be causally related to cytotoxicity occurring at an average depth of 40-50 micron into the human epidermis.

Cell Line↗
PubMed1987

Rapidly occurring DNA excision repair events determine the biological expression of u.v.-induced damage in human cells.

We have developed a new assay which allows us to monitor the rates of repair of potentially lethal damage in u.v. (254 nm)-irradiated normal human skin fibroblasts. Using this assay we have shown that, in non-dividing cells, the majority of biologically effective excision repair is completed within 4 h following irradiation with low fluences of u.v. (1.5-6.0 J/m2). During this time, non-dividing cells removed only approximately 20% of the pyrimidine dimers induced in DNA by a u.v. fluence of 3.0 J/m2 as measured by the loss of u.v.-endonuclease-sensitive sites under identical repair conditions. The rates of repair of potentially lethal damage were also found to be independent of u.v. fluence over the range 1.5-6.0 J/m2 in non-dividing cells. In contrast, in cells irradiated in exponential growth with 1.5 J/m2, the rate of biologically effective repair was comparable with that observed in non-dividing cells but the efficiency of the repair process declined progressively with increase in u.v. fluence from 1.5 to 6.0 J/m2. Our data support the concept that the biological recovery of u.v.-irradiated cells depends on the preferential repair of damage in functionally important domains in the genome.

Aphidicolin↗
PubMed1986

Repair of near-ultraviolet (365 nm)-induced strand breaks in Escherichia coli DNA. The role of the polA and recA gene products.

The action of near-ultraviolet (UV-365 nm) radiation in cellular inactivation (biological measurements) and induction and repair of DNA strand breaks (physical measurements) were studied in a repair-proficient strain and in polA-, recA-, uvrA-, and polA uvrA-deficient strains of Escherichia coli K-12. The induction of breaks in the polA and polA uvrA strains was linear with dose (4.0 and 3.7 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively). However, in the recA-, uvrA-, and repair-proficient strains, there was an initial lag in break induction at low doses and then a linear induction of breaks at higher doses with rates of 4.6, 2.8, and 3.2 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively. We interpret these strain differences as indicating simultaneous induction and repair of breaks in polymerase 1 (polA)-proficient strains under the 0 degrees C, M9 buffer irradiation conditions that, for maximum efficiency, require both the polA and recA gene products. Strand-break rejoining also occurred at 30 degrees C in complete growth medium. We propose that at least three (and possibly four) distinct types of pathways can act to reduce the levels of 365-nm radiation-induced strand breaks. A quantitative comparison of the number of breaks remaining with the number of lethal events remaining after repair in complete medium at 30 degrees C showed that between one and three breaks remain per lethal event in the wild-type and recA strains, whereas in the polA strain one order of magnitude more breaks were induced.

DNA Polymerase II↗
PubMed1986

Quantitative differences in host cell reactivation of ultraviolet-damaged virus in human skin fibroblasts and epidermal keratinocytes cultured from the same foreskin biopsy.

Repair efficiency of cultured cells may be estimated by measuring the ability of a particular cell type to support virus damaged by an appropriate agent. In this study we have compared the inactivation of ultraviolet (254 nm)-damaged herpes simplex virus in human fibroblast and epidermal keratinocyte cell lines derived from the same foreskin biopsy and found the epithelial cells to be a factor of 3 times less efficient in supporting the damaged virus. The two different cell types show comparable ultraviolet inactivation of clone-forming ability, indicating that the difference is specific to viral host cell reactivation. This study required the development of a quantitative infectious centers assay for the measurement of viral titer in human epithelial cells, a system which may be of more general application in studies of potential human carcinogens.

Adult↗
PubMed1985

A common pathway for protection of bacteria against damage by solar UVA (334 nm, 365 nm) and an oxidising agent (H2O2).

Pre-exposure of growing bacterial populations to low concentrations of hydrogen peroxide (H2O2) protects a repair-proficient strain of Escherichia coli (AB1157) very strongly and a rec A strain (AB2463) to a lesser extent from the lethal action of subsequent exposure to 5 mM H2O2 in buffer. The conditioning procedure also protects AB1157 and AB2463 from the toxic effects of UVA (334 nm, 365 nm) radiation but not UVB (313 nm) or UVC (254 nm) radiations. Pretreatment of growing AB1157 with low fluences of UVA (365 nm) radiation leads to the induction of resistance to H2O2, an effect which apparently requires protein synthesis. As in a previous report, the treatment of growing populations with low concentrations of H2O2 enhanced the resistance of such populations to H2O2 challenge in the growth medium. However, when H2O2 (+ Cu2+)-treated bacteriophage were subsequently infected into AB1157 under optimal inducing conditions, their resistance was not enhanced relative to infection into untreated bacteria. We conclude that the primary mechanism for the inducible effects observed could be the induction of H2O2 scavenging activity by low concentrations of H2O2 either introduced into the growth medium directly or produced by low fluences of UVA irradiation.

Bacteriophage lambda↗