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Biomedical subjects

M H Green

Publications and source records attributed to M H Green.

At least 37 records · Page 2Linked to original sources
PubMed1999

Frequencies of hprt(-) mutations and bcl-2 translocations in circulating human lymphocytes are correlated with United Kingdom sunlight records.

Between 1983 and 1995 we have monitored human populations for evidence of exposure to environmental mutagens, taking blood samples to measure hprt(-) mutant frequency in T cells and more recently bcl-2 t(14:18) translocation frequency in B cells. We have now analysed data from 785 assays on 448 blood samples from 308 normal subjects and find that there is a highly significant statistical correlation between hprt(-) mutant frequency and the sunlight record for the 3 weeks prior to taking the blood sample. We discuss the weaknesses in retrospective studies of this nature and the possibility of spurious epidemiological correlations that may result. More controlled experiments can be envisaged that would give a firmer basis to the statistical associations observed. hprt(-) mutations in T cells show little evidence of a UV fingerprint, so that the correlation may be due to immunomodulation rather than mutation. We also find a correlation between the sunlight record and bcl-2 translocation. This translocation is found at a low frequency in the B cells of many normal subjects and is the commonest translocation observed in non-Hodgkin's lymphoma. Our results strengthen the case for a link between sunlight and this increasingly common cancer.

Databases, Factual↗
PubMed1999

Possible effects of sunlight on human lymphocytes.

The human population is exposed to both the ultraviolet A (UVA) and B (UVB) regions of the solar spectrum. UVB induces mainly dipyrimidine photoproducts in DNA by a direct photochemical mechanism, whereas UVA is absorbed by other cellular constituents and induces mainly oxidative damage indirectly. The proportions of the different dipyrimidine photoproducts, and the ratio of dipyrimidine to oxidative damage depend on the exact spectral output of a UV source. Irradiation of human epidermal keratinocytes induces release of cytokines, with cyclobutane pyrimidine dimers playing a significant role in the process. These cytokines may then modulate the activity of cells of the immune system. Freshly isolated human lymphocytes are exquisitely sensitive to UVB irradiation, because of their low deoxyribonucleotide pools. They also have a separate defect in removal of cyclobutane pyrimidine dimers from their DNA. We have observed that frequencies of mutations at the hprt locus in human T-lymphocytes and translocations involving the bcl2 locus in B-lymphocytes appear to be associated with sunlight levels over the period before the blood sample was taken. This may be an indirect cytokine-mediated effect, and may be relevant to the possible link between non-Hodgkin's lymphoma and sunlight. On the other hand, sunlight can have beneficial effects, and may protect against autoimmune diseases including type I diabetes and multiple sclerosis.

Cell Transformation, Neoplastic↗
PubMed1999

Glucose kinetic responses to protein supplementation and exogenous somatotropin in late gestation dairy cows.

Glucose kinetics were measured in late gestation multiparous Holstein dairy cows fed diets with different amounts of dietary crude protein (13.3 vs. 17.8%), with and without exogenous somatotropin. The trial was conducted as a completely randomized design; 35 cows were used in the final analysis. Kinetic measurements were made using the single injection technique with uniformly labeled 13C-labeled glucose. A diet that contained 17.8% crude protein appeared to increase glucose utilization without a corresponding increase in supply. The evidence was an increased rate of glucose disposal. In contrast, exogenous bovine somatotropin (Posilac, Monsanto Co., St. Louis, MO) appeared to enhance glucose conservation, as indicated by a tendency for a decreased fractional catabolic rate and an increased glucose pool size. Somatotropin appeared to modify glucose metabolism in a pattern favorable for supporting terminal fetal development and lactogenesis and for maintaining or enhancing maternal glycemia. The latter pattern could have implications for improving the health of periparturient dairy cows.

Animals↗
PubMed1999

In search of an "Authentic" women's medicine: the strange fates of Trota of Salerno and Hildegard of Bingen.

Despite centuries of debate about the medieval writers Trota and Hildegard, there still remain widely disparate views of them in both popular and scholarly discourses. Their alternate dismissal or romanticization is not due to a simple contest between antifeminist and feminist tendencies. Rather, issues of gender have intersected in varying ways with other agendas (intellectual, nationalist, etc.). Recent philological researches have helped not only to clarify why these earlier interpretations were created in the first place, but also to raise our understanding of these women and their work to a new, higher level.

Germany↗
PubMed1998

c-Myc is a major mediator of the synergistic growth inhibitory effects of retinoic acid and interferon in breast cancer cells.

The molecular signaling events involved in the inhibition of breast cancer cell growth by retinoic acid and interferon-alpha were investigated. All-trans-retinoic acid and interferon-alpha acted synergistically to inhibit growth of both the estrogen receptor-positive breast cancer cell line MCF-7 and the estrogen receptor-negative line BT-20. In MCF-7 cells, all-trans-retinoic acid potentiated the effects of interferon-alpha by up-regulating the expression of the RNA-dependent protein kinase (PKR). Consequently, the synergism between all-trans-retinoic acid and interferon-alpha down-regulated the expression of c-Myc, but not its functional partner, Max. Transfection of MCF-7 cells with a dominant-negative mutant of PKR relieved c-Myc down-regulation and cell growth inhibition, indicating that PKR is directly involved in c-Myc down-regulation and that c-Myc down-regulation is responsible for the inhibition of cell growth. Corresponding with c-Myc down-regulation, c-Myc.Max heterodimers bound to their consensus DNA sequence were undetectable in cells treated with all-trans-retinoic acid and interferon-alpha, indicating diminished c-Myc functionality. When c-Myc was overexpressed ectopically via a c-Myc expression vector, MCF-7 cells became resistant to growth inhibition by all-trans-retinoic acid plus interferon-alpha. These experiments define the following pathway as a major pathway in the synergistic growth inhibition of MCF-7 cells by all-trans-retinoic acid plus interferon-alpha: all-trans-retinoic acid + interferon-alpha --> upward arrow double-stranded RNA-dependent protein kinase --> downward arrow c-Myc --> cell growth inhibition.

Antineoplastic Agents↗
PubMed1998

Growth factor protection against cytokine-induced apoptosis in neonatal rat islets of Langerhans: role of Fas.

Treatment of neonatal rat islets of Langerhans with combined cytokines (interleukin-1beta 10(-10) M, tumour necrosis factor-alpha 10(-10) M, interferon-gamma 5 U/ml) led to extensive cell death, which was potentiated by Fas activation with the anti-Fas cytolytic antibody JO2. Pre-treatment with insulin (25 ng/ml) or insulin-like growth factor-1 (10(-8)M) gave only partial protection against cell killing, but prevented the Fas-mediated component. In the absence of cytokine treatment, Fas-mediated killing was not observed.

Animals↗
PubMed1998

Factors affecting the DNA damaging activity of superoxide and nitric oxide.

Nitric oxide and superoxide are formed endogenously and can react with each other and with other molecules to form a range of secondary and tertiary products. Some of these (e.g., peroxynitrite) are potent DNA-damaging agents and others (e.g., S-nitrosoglutathione) can act as reservoirs of the reactive species. Although the chemistry of these processes is now becoming understood, the question of which products are significant in vivo is not necessarily clear. To investigate these processes we have developed a cell-free version of the Comet assay, where the DNA from isolated nuclei is treated in agar on a microscope slide, following lysis. This offers an exceptionally sensitive assay for strand breakage in free DNA. Despite being present as a scavenger in the cell at millimolar levels, glutathione can act as a DNA-damaging pro-oxidant. Under appropriate conditions, glutathione-mediated damage is suppressed by superoxide dismutase and we suggest that superoxide may be a direct damaging agent, whose activity can be masked because of the involvement of superoxide in indirect mediation of damage or because of concomitant presence of hydroxyl radical.

Animals↗
PubMed1998

Use of the Comet assay to investigate the role of superoxide in glutathione-induced DNA damage.

Although glutathione is an important scavenging molecule within the cell, it can also act as a pro-oxidant and at biological concentrations (1 mM) can induce DNA damage. We have used a sensitive cell-free Comet assay for DNA strand breakage to investigate this damage and to try to determine the active species involved. We show a substantial protection against glutathione-mediated DNA damage by superoxide dismutase (200 U/ml) and complete protection by combined superoxide dismutase and catalase. Damage is also prevented by EDTA but only at 100 mM and is not prevented by the chelating agent diethylenetriamine-pentaacetic acid (100 microM). Although superoxide is known to potentiate DNA damage by other reactive species, none of these indirect mechanisms seem to account for our results and it is possible that superoxide may damage DNA directly. Under the same experimental conditions, S-nitrosoglutathione requires ultraviolet A photolysis to cause DNA strand breakage and superoxide dismutase increases the level of this damage. When intact human lymphocytes are incubated with glutathione (1 mM) in phosphate buffer, DNA damage is also observed, but in this case it is completely preventable by catalase, with no protective effect of superoxide dismutase. Since cellular scavenging systems are not completely protective against reactive species formed from autooxidation of extracellular glutathione and since glutathione and oxygen are ubiquitously present within cells, our results imply that cells may have a mechanism of preventing autooxidation, rather than simply relying on scavenging the reactive species formula.

Catalase↗
PubMed1998

Insulin secretion, DNA damage, and apoptosis in human and rat islets of Langerhans following exposure to nitric oxide, peroxynitrite, and cytokines.

Cytokine-induced damage may contribute to destruction of insulin-secreting beta-cells in islets of Langerhans during autoimmune diabetes. There is considerable controversy (i) whether human and rat islets respond differently to cytokines, (ii) the extent to which cytokine damage is mediated by induction of nitric oxide formation, and (iii) whether the effects of nitric oxide on islets can be distinguished from those of reactive oxygen species or peroxynitrite. We have analyzed rat and human islet responses in parallel, 48 h after exposure to the nitric oxide donor S-nitrosoglutathione, the mixed donor 3-morpholinosydnonimine, hypoxanthine/xanthine oxidase, peroxynitrite, and combined cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma). Insulin secretory response to glucose, insulin content, DNA strand breakage, and early-to-late stage apoptosis were recorded in each experiment. Rat islet insulin secretion was reduced by S-nitrosoglutathione or combined cytokines, but unexpectedly increased by peroxynitrite or hypoxanthine/xanthine oxidase. Effects on human islet insulin secretion were small; cytokines and S-nitrosoglutathione decreased insulin content. Both rat and human islets showed significant and similar levels of DNA damage following all treatments. Apoptosis in neonatal rat islets was increased by every treatment, but was at a low rate in adult rat or human islets and only achieved significance with cytokine treatment of human islets. All cytokine responses were blocked by an arginine analogue. We conclude: (i) Reactive oxygen species increased and nitric oxide decreased insulin secretory responsiveness in rat islets. (ii) Species differences lie mainly in responses to cytokines, applied at a lower dose and shorter time than in most studies of human islets. (iii) Cytokine effects were nitric oxide driven; neither reactive oxygen species nor peroxynitrite reproduced cytokine effects. (iv) Rat and human islets showed equal susceptibility to DNA damage. (v) Apoptosis was not the preferred death pathway in adult islets. (vi) We have found no evidence of human donor variation in the pattern of response to these treatments.

Adult↗
PubMed1998

Use of model-based compartmental analysis to study effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on vitamin A kinetics in rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic, widespread environmental contaminant that has dramatic adverse effects on the metabolism of vitamin A. We used model-based compartmental analysis to investigate sites and quantitative impacts of TCDD on vitamin A kinetics in rats given on oral loading dose of TCDD in oil (3.5 micrograms/kg) followed by weekly maintenance doses (0.7 microgram/kg) or oil only. [3H]Retinol in its plasma transport complex (experiment 1) of lymph containing chylomicrons labeled mainly with [3H]retinyl esters (experiment 2) were administered i.v., and tracer kinetics in plasma, liver, carcass, urine, and feces were measured for up to 42 days. TCDD treatment caused significant reductions in liver vitamin A levels and significant changes in tracer kinetics and tracer excretion. A four-compartment model was used to fit tracer data for experiment 1; for experiment 2, compartments were added to describe the metabolism of newly absorbed vitamin A. The compartmental models predict that TCDD caused a slight delay in plasma clearance (via an increased recycling to plasma), and in liver processing, of chylomicron-derived vitamin A. Models for both experiments predict that TCDD exposure did not affect the fractional uptake of plasma retinol from the rapidly turning-over extravascular pool, but it doubled the fractional transfer of recycled retinol from slowly turning-over pools of vitamin A to plasma. The residence time for vitamin A was reduced by 70% in TCDD-treated rats, transfer into urine and feces was tripled, and vitamin A utilization rates were significantly increased. Since our results do not indicate that retinol esterification is inhibited, we hypothesize that some of the significant effects of TCDD on vitamin A metabolism result from increased catabolism and mobilization of vitamin A from slowly turning-over pools (especially the liver).

Animals↗
PubMed1998

Development of a compartmental model describing the dynamics of vitamin A metabolism in men.

Model-based compartmental analysis was used with the Simulation, Analysis and Modeling (SAAM) computer programs to analyze data on plasma retinoid kinetics in adult male subjects for 7 d after a single oral dose of 105 mumol of [8,9,19-13C]retinyl palmitate. We present here the data for one subject and discuss in detail the steps taken to develop a physiologically-based compartmental model that describes the dynamic behavior of plasma retinyl esters, [12C]retinol, [8,9,19-13C]retinol, and the sum of [12C] and [13C]retinol. First an absorption model was developed to fit data on the plasma appearance and disappearance of retinyl esters; this was used as input in development of models for labeled and unlabeled retinol. The large oral load of labeled vitamin A perturbed the unlabeled tracee system, and thus parallel models for tracer and tracee were developed; and a time-variant fractional transfer coefficient was incorporated into the tracee model. Following the absorption model, four-compartment models were developed to describe the dynamics of both labeled and unlabeled retinol. These models predict that, in spite of the large vitamin A load, the absorption efficiency was 34%; hepatic (presumably parenchymal cell) processing of the absorbed dose was essentially complete by 24 h; and, by 7 days, approximately 80% of the absorbed dose was in a compartment that presumably represents stored liver retinyl esters. The model also predicts that approximately 50 mumol of retinol passed through the plasma each day, compared to an estimated utilization rate of 4 mumol/day. This project provides unique and important information about whole-body vitamin A dynamic in humans, and presents approaches to specific modeling issues that may be encountered by others.

Adult↗
PubMed1998

Induction of interleukin-6 production by ultraviolet radiation in normal human epidermal keratinocytes and in a human keratinocyte cell line is mediated by DNA damage.

The sunburn reaction is the most common consequence of human exposure to ultraviolet radiation (UVR), and is mediated at least in part by interleukin-6 (IL-6). The aim of this study was to determine if DNA is a major chromophore involved in the induction of IL-6 following UV irradiation of a human epidermoid carcinoma cell line (KB), and of normal human epidermal keratinocytes. We first confirmed that IL-6 release was associated with enhanced levels of IL-6 mRNA transcripts. The wavelength dependence for IL-6 release was then investigated by irradiating the cells at defined wavelengths (254, 302, 313, 334, and 365 nm) with a monochromator. The maximum effect on IL-6 release was observed at 254 nm with only low levels of induction observed at wavelengths above 313 nm. The wavelength dependence for UV-induced IL-6 release was similar to that for DNA absorption or for the induction of cyclobutane pyrimidine dimers (CPD). To determine whether UV-induced DNA damage mediated IL-6 secretion, the role of CPD was investigated by treating keratinocytes with photosomes (photolyase encapsulated in liposomes) followed by photoreactivating light. This photoreversal procedure led to a reduction in the levels of the UVC-induced secretion of IL-6, which in normal human keratinocytes was unambiguously associated with repair of CPD. We conclude that the release of IL-6 from human keratinocytes following short-wave UVC and UVB irradiation is mediated by DNA damage and that CPD play an important role in this process.

Carcinoma, Squamous Cell↗
PubMed1998

Plasma retinol is a major determinant of vitamin A utilization in rats.

We studied relationships among vitamin A intake, liver levels of vitamin A, plasma retinol concentrations and the irreversible utilization of vitamin A. To supplement existing data, we first used model-based compartmental analysis to determine vitamin A utilization and other kinetic parameters in male Sprague-Dawley rats that had adequate liver vitamin A stores ( approximately 9000 nmol) and were fed a diet containing low levels of vitamin A. Plasma retinol kinetics were monitored for 43 d after administration of [3H]retinol-labeled plasma to rats consuming approximately 23 (Group 1, n = 6) or approximately 4.2 (Group 2, n = 6) nmol vitamin A/d. Data for plasma tracer vs. time and for tracer lost irreversibly by the end of the experiment, were fit to a three-compartment model in which plasma retinol exchanges with vitamin A in two kinetically distinct extravascular compartments. Irreversible utilization rates ( approximately 41 nmol/d) were similar to those for rats that are in vitamin A balance, suggesting that, when liver vitamin A stores are adequate, utilization rate is not decreased to compensate for a low vitamin A intake. Multiple linear regression analysis was then used to relate these and previously collected data (total, 62 rats) on vitamin A intake (4. 2-49 nmol/d), plasma retinol concentration (1.4-2.5 micromol/L) and liver vitamin A level (1.2-11,000 nmol) to vitamin A utilization (disposal rate, 4.2-68 nmol/d). A significant relationship (R2(adj) = 0.93) was found for the equation [disposal rate (nmol/d) = -0.720 (nmol/d) + 0.844 (d-1).(plasma retinol; nmol) + 0.00139 (d-1).(liver vitamin A; nmol) + 0.220.(vitamin A intake; nmol/d)]. Plasma retinol accounted for 92% of the variability in disposal rate (vs. 5% for liver vitamin A and 3% for vitamin A intake). We conclude that plasma retinol is a main determinant of the irreversible utilization of vitamin A in rats with low to moderate vitamin A intake.

Animals↗
PubMed1998

Plasma thyroid hormone kinetics are altered in iron-deficient rats.

Iron deficiency anemia is associated with lower plasma thyroid hormone concentrations in rodents and, in some studies, in humans. The objective of this project was to determine if plasma triiodothyronine (T3) and thyroxine (T4) kinetics were affected by iron deficiency. Studies were done at a near-thermoneutral temperature (30 degrees C), and a cool environmental temperature (15 degrees C), to determine plasma T3 and T4 kinetics as a function of dietary iron intake and environmental need for the hormones. Weanling male Sprague-Dawley rats were fed either a low Fe diet [iron-deficient group (ID), <5 microg/g Fe] or a control diet [control group (CN), 35 microg/g Fe] at each temperature for 7 wk before the tracer kinetic studies. An additional ID group receiving exogenous thyroid hormone replacement was also used at the cooler temperature. For T4, the disposal rate was >60% lower (89 +/- 6 vs. 256 +/- 53 pmol/h, P < 0.001) in ID rats than in controls at 30 degrees C, and approximately 40% lower (192 +/- 27 vs. 372 +/- 26 pmol/h, P < 0.01) in ID rats at 15 degrees C. Exogenous T4 replacement in a cohort of ID rats at 15 degrees C normalized the T4 concentration and the disposal rate. For T3, the disposal rate was significantly lower in ID rats in a cool environment (92 +/- 11 vs. 129 +/- 11 pmol/h, P < 0.01); thyroxine replacement again normalized the T3 disposal rate (126 +/- 12 pmol/h). Neither liver nor brown fat thyroxine 5'-deiodinase activities were sufficiently different to explain the lower T3 disposal rates in iron deficiency. Thus, plasma thyroid hormone kinetics in iron deficiency anemia are corrected by simply providing more thyroxine. This suggests a central regulatory defect as the primary lesion and not peripheral alterations.

Anemia, Iron-Deficiency↗
PubMed1998

Repair of cyclobutane pyrimidine dimers in unstimulated human mononuclear cells is deficient at very low fluences of ultraviolet B and is not enhanced by addition of deoxyribonucleosides.

Unstimulated human T lymphocytes are exquisitely sensitive to UVB irradiation. This hypersensitivity appears to relate to low deoxyribonucleotide pool sizes. They have also been reported to be defective in global excision of cyclobutane pyrimidine dimers, but such experiments may have been carried out at supralethal doses, where unrepaired excision breaks persist indefinitely. We use a T4 endonuclease Comet assay to show that removal of cyclobutane pyrimidine dimers is defective in the unstimulated mononuclear cell fraction (mainly T lymphocytes) even at sublethal fluences from an FS20 broad spectrum UVB lamp. Moreover, removal is not enhanced by addition of deoxyribonucleosides to the medium. Cells which are failing to remove cyclobutane pyrimidine dimers readily form fresh incision breaks in response to a second UVB fluence, indicating that they retain repair capacity and suggesting that removal of types of damage other than cyclobutane pyrimidine dimers is effective.

Adult↗
PubMed1997

Use of the comet assay to investigate possible interactions of nitric oxide and reactive oxygen species in the induction of DNA damage and inhibition of function in an insulin-secreting cell line.

We have previously used the comet assay to demonstrate that the nitric oxide donor 3-morpholinosydnonimine (SIN-1) produces DNA damage in rat islets of Langerhans and in the SV40-transformed insulin-secreting hamster cell line, HIT-T15. Damage is not prevented by the addition of superoxide dismutase (SOD). In the present study, we have compared SIN-1, which generates nitric oxide, superoxide anion and hydrogen peroxide, with two other nitric oxide donors, S-nitrosoglutathione (GSNO) and the tetra-iron-sulphur cluster nitrosyl, Roussin's black salt (RBS). We have used the comet assay as a highly sensitive method to measure DNA-damaging ability, and also measured inhibition of DNA synthesis and inhibition of insulin secretion. We have examined the effect of SOD and catalase on each of these endpoints in HIT-T15 cells following a 30-min exposure to the compounds (24 h for DNA synthesis). All compounds produced a significant dose-dependent increase in strand-breakage formation and all inhibited DNA synthesis and glucose-stimulated insulin secretion. RBS was the most potent. SOD did not reduce the responses observed with any of the compounds. Catalase largely prevented DNA strand breakage, inhibition of DNA synthesis and inhibition of insulin secretion by SIN-1, but had no effect on responses to GSNO or RBS. Addition of SOD together with catalase gave no greater protection against SIN-1 than catalase alone. The nitric oxide and superoxide anion produced by SIN-1 are though to combine to form highly reactive peroxynitrite. In addition, H2O2 may be formed in the presence of SIN-1 and may form hydroxyl radical in the presence of a transition metal, such as Fe2+. It appears that in insulin-secreting cells, the effects of SIN-1 are largely mediated by this latter mechanism. In contrast, GSNO and RBS appear to act by a different mechanism, not overtly involving reactive oxygen species. GSNO and H2O2 show no significant interaction in the induction of DNA strand breaks. Both nitric oxide and H2O2 are effective, directly or indirectly, as DNA strand-breaking agents, inhibitors of DNA synthesis and inhibitors of insulin secretion.

Animals↗
PubMed1997

Correlated mutagenesis of bcl2 and hprt loci in blood lymphocytes.

In vivo measurement of human somatic mutations may be a valuable biodosimeter of exposure to carcinogens and of cancer risk. We have surveyed translocations at the bcl2 locus in B lymphocytes, and mutations at hprt in T lymphocytes, in 120 individuals with varying exposure to radon and cigarette smoke. bcl2 t(14:18) translocation is the commonest chromosomal alteration observed in non-Hodgkins lymphoma (NHL). We observed a significantly larger range of bcl2 translocation frequency (range: 0-372 x 10(-6), median: 1.9 x 10(-6)) than of hprt mutation frequency (range: 0-76.4 x 10(-6), median: 11.1 x 10(-6)), which is likely the result of clonal proliferation of deathless B cell mutants. We observed that the frequencies of these two distinct lymphocytic mutations are significantly correlated. Although some of the correlated variation is explained by age, a significant correlation of bcl2 mutagenesis persists after age adjustment. Correlated mutagenesis at distinct loci in distinct cell types could be explained by the existence of a mutator phenotype or by variation in exposure to environmental mutagens. NHL is commoner in men than in women, and our data indicate a trend toward higher bcl2 mutagenesis in males than females. There is mounting epidemiological evidence for a worldwide increase in NHL, which may have an environmental basis; molecular epidemiological analysis of bcl2 mutagenesis in exposed populations might be especially relevant to the identification of putative environmental causes. Given the relative ease of the bcl2 assay versus the hprt assay, and the consistency with which data are reproduced from laboratory to laboratory, it is likely that the bcl2 assay will be soon added to the array of assays used in human mutational surveillance.

Adolescent↗