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Biomedical subjects

Y A Barnett

Publications and source records attributed to Y A Barnett.

At least 19 recordsLinked to original sources
PubMed1998

DNA damage and mutation: contributors to the age-related alterations in T cell-mediated immune responses?

The genetic material of our cells is susceptible to damage by a wide variety of extrinsic and intrinsic entities. The amount of genetic damage accumulated in vivo will depend upon an individual's ability to defend against and/or repair DNA damage. T cells in vivo have been shown to accumulate DNA damage and mutations over time. The accumulation of such genetic damage will occur in T cells possessing a 'naive' or a 'memory' phenotype. Since T cells are required to undergo extensive clonal expansion upon antigenic stimulation, DNA damage and mutations may result in: a failure of T cells to proliferate, because of DNA damage-mediated cell cycle arrest; decreased rates of proliferation, as a consequence of selection in vivo against cells carrying certain mutations and/or apoptosis, triggered by critical levels of DNA damage. Thus, when T cells, containing critical levels of genetic damage, are required to undergo rapid clonal expansion in the presence of antigen, insufficient numbers of T cells may be produced and so the immune response would be sub-optimal. In this paper the possible contribution of DNA damage and/or mutation to the age-related alterations in T cell-mediated immune responses will be discussed.

Aging↗
PubMed1997

In vivo antioxidant status, DNA damage, mutation and DNA repair capacity in cultured lymphocytes from healthy 75- to 80-year-old humans.

The accumulation of damage to cellular biomolecules, including DNA, over time may play a significant role in the aetiology of the ageing process. We have previously quantified DNA damage and mutation within cultured lymphocytes from healthy human male subjects in three different age groups (35-39, 50-54 and 65-69 years). The results of that study showed an age-related increase in DNA damage and mutations in lymphocytes. In addition, an age-related decrease in the capacity of the lymphocytes to repair H2O2-induced DNA damage was found. In this article, we report the findings of an extension to the earlier study. Thirty-one generally healthy male and female subjects between the ages of 75 and 80 years were recruited. Using a number of bioassays, we were able to determine; basal levels of DNA damage (for 18 subjects) and mutant frequency at the hypoxanthine phosphoribosyltransferase (hprt) gene locus (for 16 subjects) within cultured lymphocytes. In addition, in vivo antioxidant status (for all study subjects) and the capacity of lymphocytes to repair H2O2-induced DNA damage (for 18 subjects) were also assessed. The results obtained showed: that the mean basal level of DNA damage in lymphocytes from subjects in the 75- to 80-year age group (12.6 +/- 4.7%) was similar to that of the 35- to 39-year age group (13.3 +/- 3.3%), p = 0.42 (Mann-Whitney); there was no significant difference between log mean mutant frequency at the hprt gene locus in lymphocytes from the 75- to 80-year age group (0.31 +/- 0.33) compared to that observed in the 35- to 39-year age group (0.24 +/- 0.21; Student's t-test, t = 0.68, p > 0.05). Levels of the antioxidants glutathione peroxidase (GPx EC 1.11.1.9), catalase (CAT; EC 1.11.1.6) and caeruloplasmin (CPL; EC 1.16.3.1) were significantly elevated in the 75- to 80-year age group, compared to the 35- to 39-, 50- to 54- and 65- to 69-year age groups. Levels of bilirubin (BR) were reduced in the 75- to 80-year age group, the decrease being contributed by the female subjects. No differences in levels of superoxide dismutase (SOD; EC 1.15.1.1) or uric acid (UA) were found between the 4 age groups. Following treatment of lymphocytes with H2O2, we did not find any difference in the susceptibility of lymphocytes to DNA damage in the 75- to 80-year age group, compared to the other age groups. The DNA repair capacity in lymphocytes from individuals in the 75- to 80-year age group was similar to that of the 35- to 39-year age group, for all time points assessed. These results highlight the importance of DNA repair processes and antioxidant defence systems for maintaining genomic stability in vivo.

Aged↗
PubMed1995

Quantification of DNA damage and repair in amino acid auxotrophs and UV-sensitive mutants of Aspergillus nidulans using an ELISA.

An ELISA used to investigate DNA repair in mammalian cells has been adapted to investigate mutagen-induced DNA damage and repair in protoplasts of Aspergillus nidulans. The assay shows a reduced rate of repair of DNA damage in methionine and arginine auxotrophs (methG and argB), which were shown previously to be hypersensitive to UV radiation and chemical mutagens. The assay also showed a considerably reduced ability to repair mutagen-induced damage in the uv-sensitive mutants uvsB and uvsH. The increased sensitivity of amino acids auxotrophs to mutagens is, therefore, correlated with a reduced capacity to repair mutagen-induced DNA damage.

4-Nitroquinoline-1-oxide↗
PubMed1995

An investigation of the cytotoxic and mutagenic potential of low intensity laser irradiation in Friend erythroleukaemia cells.

The purpose of this study was to investigate the cytotoxic and genotoxic potential of low intensity laser irradiation (660 nm, 12 mW, 5 kHz) on mammalian cells. Thymidine kinase (TK)-positive and TK-deficient Friend erythroleukaemia (FEL) cells, clone 707 and subclone 707BUF respectively, were used in this investigation. Following irradiation of exponentially growing cells in suspension at doses of 2 and 20 J/cm2 a number of sensitive bioassays were used to facilitate the detection of laser-induced mutations, DNA damage and cell killing. Mutations were assessed by the examination of chromosome spreads, the determination of micronucleus frequency and by the determination of the mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. DNA damage was quantified using a sensitive ELISA. The cytotoxic effect of laser irradiation was assessed using a cloning assay. The results of this investigation did not show any significant increase in mutation frequency, DNA damage or cell survival in the laser-irradiated cells, compared to sham-irradiated controls. The lack of any demonstrable cytotoxic and genotoxic effects of low intensity laser irradiation on mammalian cells in culture would support it as being a safe modality for clinical use.

Cell Line↗
PubMed1995

An investigation of antioxidant status, DNA repair capacity and mutation as a function of age in humans.

We are constantly exposed, throughout life, to a wide variety of extrinsic and intrinsic agents which have the potential to damage cellular biomolecules, including DNA. Imperfections in cellular defence systems which protect against the fixation of DNA damage can lead to an accumulation of mutations which on their own, or in combination with other age-related changes, may contribute to ageing and the development of age-related pathologies. We have previously reported an increase in frequency of mutation with age in human lymphocytes taken from healthy males in the age groups, 35-39, 50-54 and 65-69 years. In this article we report on the findings of a recent study which was designed to assess whether the age-related increase in frequency of mutation was due to a decreased efficacy of the defence systems against ROS-induced DNA damage, namely antioxidant status and DNA repair processes, in the same study subjects. In vivo antioxidant status was assessed in each of the study subjects by measuring blood levels of; superoxide dismutase (SOD; EC 1.15.1.1), glutathione peroxidase (GPx; EC 1.11.1.9), catalase (EC 1.11.1.6), caeruloplasmin (CPL), uric acid and bilirubin. We did not find any statistically significant differences in the mean levels of these antioxidants between the three different age groups. To investigate the efficacy of DNA repair processes against ROS-induced DNA damage, an ELISA was used to quantitate DNA damage (as % single-stranded DNA; %SS-DNA) at various times following treatment of peripheral blood lymphocytes with hydrogen peroxide (H2O2). The results of this part of the study showed that in untreated lymphocytes, basal levels of %SS-DNA were significantly higher in individuals from the 65-69 years age group compared to the 35-39 years age group (p = 0.039, 0.0013; at 5% level of significance). No significant differences were found in H2O2 susceptibility with age immediately following treatment (p = 0.71, 1.00; at 5% level of significance) but a consistent and significant increase was observed in %SS-DNA remaining 90 min post-treatment in lymphocytes from subjects in the 65-69 years age group, compared to %SS-DNA present in lymphocytes from the 35-39 years age group (p = 0.013, 0.024; at 5% level of significance). The results of this study suggest that the age-related increase in frequency of mutations is not contributed to by alterations of in vivo antioxidant status with age but is by a decreased efficacy of the repair of ROS-induced DNA damage with age. The biological implications of somatic mutations in the ageing process are discussed.

Adult↗
PubMed1995

Detection of reverse transcriptase in culture medium for mammary tumour cell lines: a comparison of an established radio-labelling technique and a contemporary non-isotopic technique.

Classically, radio-label techniques have been employed to analyse biological samples for reverse transcriptase (RT) activity. More recently, however, non-isotopic kits have been developed for retroviral quantification. Nevertheless, until the present investigation it has not been known if these contemporary methods are more sensitive at detecting reverse transcriptase activity. In our study, a non-isotopic ELISA method was shown to be considerably more sensitive than the radio-label technique at detecting reverse transcriptase in growth medium used to culture the murine breast cancer cell line GR/A. Using the ELISA, less reverse transcriptase activity was demonstrated in growth medium from human mammary adenocarcinoma MCF-7 cells than the murine source. This ELISA did not detect reverse transcriptase activity from a pure source of Moloney murine leukaemia virus. In light of this, the broad applicability of this ELISA for reverse transcriptase from different viral sources must be investigated before it can be used to monitor biological supernatants for the presence of retroviruses.

Animals↗
PubMed1995

Oxidant-induced stress response in lymphoid cells.

The stress response to reactive oxygen species is an important defence system which can reduce their potential to induce biomolecule damage. In this investigation the effect of exposing Molt-3 lymphoblastoid cells or peripheral blood lymphocytes to a non-toxic dose of hydrogen peroxide (10 microM) was studied. Cellular response to a subsequent high dose of hydrogen peroxide (100-200 microM) was assessed by measurement of growth, viability, proliferation and DNA damage (lymphocytes only) and intracellular activities of the enzymes, superoxide dismutase, glutathione peroxidase and catalase (Molt-3 only). The results indicate that pretreatment of lymphocytes with 10 microM hydrogen peroxide can elicit a response which is protective against DNA damage normally inducible in these cells by subsequent exposure to toxic doses of hydrogen peroxide. It appears from the results with Molt-3 cells that altered activities of glutathione peroxidase may contribute to this enhanced resistance to hydrogen peroxide.

Cell Division↗
PubMed1994

Germinating conidiospores of Aspergillus amino acid auxotrophs are hypersensitive to heat shock, oxidative stress and DNA damage.

Germinating conidiospores (conidia) of Aspergillus nidulans amino acid-requiring strains are hypersensitive to heat, oxidative stress, UV radiation and chemical mutagens when compared with other strains. They also showed an increased mutation rate. Sensitivity to stress conditions has been correlated with an abnormal RAS/cAMP pathway in mutants of S. cerevisiae. We suggest that the RAS/cAMP pathway is defective in germinating conidia of Aspergillus amino acid auxotrophs and that this is responsible for suppressing DNA repair and conferring sensitivity to oxidative stress and heat shock.

4-Nitroquinoline-1-oxide↗
PubMed1994

An investigation of mutation as a function of age in humans.

An accumulation of mutations on their own or together with other age-related changes may contribute to aging and the development of age-related pathologies. The aim of this investigation was to assess the extent of DNA mutations as a function of age in humans. The mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl-transferase (hgprt) locus was assessed in lymphocytes isolated from male volunteers in each of three age groups (35-39, 50-54 and 65-69 years). Results show that the mean MF in the 65-69 years group was approximately twice that in the 35-39 and 50-54 years groups (4.1/10(6) cells, 1.9/10(6) cells and 1.79/10(6) cells, respectively) increasing by about 1.33% per year, after 54 years. In addition, there was an increased frequency of chromosomal aberrations in the 65-69 years group compared to the other two age groups. The results of this investigation show an increase in DNA mutations in cultured human lymphocytes with age. Factors which may influence the extent of DNA damage in human lymphocytes are discussed.

Adult↗