Nitric oxide rather than superoxide or peroxynitrite inhibits insulin secretion and causes DNA damage in HIT-T15 cells.
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Animals↗
Biomedical subjects
M H Green
Publications and source records attributed to M H Green.
Hypersensitivity of ataxia-telangiectasia fibroblasts to a nitric oxide donor.
Ataxia-telangiectasia (A-T) is a human autosomal recessive disease characterised by immunodeficiency, extreme sensitivity to ionising radiation and progressive cerebellar ataxia. The defective gene has recently been cloned and is a member of the phosphatidylinositol 3-kinase family. We have investigated the possibility that the neurodegeneration in A-T might be induced by an endogenously formed mutagen causing radiation-like damage. Nitric oxide is known to be formed in the cerebellum and we present evidence that A-T fibroblasts are hypersensitive to killing by the nitric oxide donor S-nitrosoglutathione (GSNO), as are fibroblasts from a radiosensitive individual without ataxia. Killing was determined as loss of colony forming ability. GSNO induces dose-dependent DNA strand breakage, but to no greater extent in A-T fibroblasts. Breakdown of GSNO to nitrite and nitrate appears to occur to the same extent in both normal and A-T fibroblasts. Cell killing by GSNO appears to be associated in both types of cell with formation of nitrite, rather than nitrate, as the ultimate oxidation product of nitric oxide.
Ataxia Telangiectasia↗
N-3 fatty acids and chylomicron metabolism in the rat.
Dietary n-3 fatty acids (FAs) reduce postprandial triacylglycerol concentrations in humans by unknown mechanisms. Our goals were to reproduce this phenomenon in the rat, and then to determine the mechanism. In an oral fat tolerance study two groups of rats were fed diets containing 2.1% ethyl esters of n-3 FA or olive oil for 2 weeks. After gavaging with emulsified soybean oil, the postprandial chylomicron triacylglycerol levels in the n-3 FA group were reduced by 40% (P < 0.05). The hypothesis that n-3 FA feeding reduced chylomicron production/secretion from the gut was tested by blocking chylomicron removal with Triton WR1339 before gavaging the rats with the fat load. This completely eliminated the hypochylomicronemic effect suggesting that chylomicron input was not inhibited by n-3 FAs. Chylomicron clearance was studied by injecting chylomicrons containing radioactive retinyl esters and triacylglycerol into rats from both groups. Pre-feeding with n-3 FAs accelerated the removal of chylomicron triacylglycerol and retinyl esters from the plasma with significantly lower fractions of dose remaining at 2, 4, and 8 min post-injection for both tracers. These findings suggest than n-3 FAs reduce postprandial chylomicronemia in the rat by accelerating chylomicron lipid clearance.
Animals↗
Biomonitoring of possible human exposure to environmental genotoxic chemicals: lessons from a study following the wreck of the oil tanker Braer.
In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.
Accidents, Occupational↗
Induction of mutagenic DNA damage in human fibroblasts after exposure to artificial tanning lamps.
There is increasing concern about the adverse health effects associated with the use of sunbeds, particularly with respect to skin photocarcinogenesis. The induction of mutagenic DNA damage is a prerequisite for the development of skin tumours, and it is well established that direct types of damage such as cyclobutane pyrimidine dimers (CPDs) give rise to mutations in tumour suppressor genes and oncogenes. In addition, ultraviolet radiation may induce indirect types of DNA damage, including oxidative products, which are also potentially mutagenic. By using specific DNA repair enzymes (T4 endonuclease V and endonuclease III) and the comet assay we have been able to detect the induction of CPDs, oxidized or hydrated pyrimidine bases and single-strand breaks in cultured human fibroblasts (MRC-5) after exposure for between 15 s and 20 min on two different commercial sunbeds containing Philips 'Performance' 100W-R or Philips TL80W/10R lamps. The ratio of endonuclease III to T4 endonuclease V sensitive sites varied substantially between the two lamps and was 3.3% and 18%, respectively. The sunbed containing the 'Performance' 100W-R lamps was as potent at inducing CPDs as was natural sunlight in fine weather. These results establish that commercial tanning lamps produce the types of DNA damage associated with photocarcinogenesis in human cells, and complement epidemiological evidence indicating the potential risk of using sunbeds.
Beauty Culture↗
Inhibition of RNA and DNA synthesis in UV-irradiated normal human fibroblasts is correlated with pyrimidine (6-4) pyrimidone photoproduct formation.
UV-irradiation of living cells results in an inhibition of RNA and DNA synthesis. The purpose of this study was to determine whether specific photoproducts or the total combined yield of lesions were responsible for these effects. Asynchronously dividing human fibroblasts from normal donors were irradiated with UVC (254 nm), broad spectrum UVB (290-320 + nm, Westinghouse FS20 lamp) or narrow spectrum UVB (310-315 nm, Philips TL01 lamp) at fluences which induce known yields of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts or Dewar isomers. DNA synthesis was approximately 3-4 times more sensitive to both UVC and UVB irradiation than RNA synthesis. The immediate inhibition of RNA and DNA synthesis was correlated with (6-4) rather than overall photoproduct formation suggesting that the (6-4) photoproduct is the mediator of these inhibitory effects. In support of this suggestion we found that photoreactivation of cells cultured from the marsupial, mouse Sminthopsis crassicaudata, resulted in removal of 70% of pyrimidine dimers from the overall genome, but had only a slight effect on the recovery of RNA synthesis.
Animals↗
Nitric oxide donors decrease the function and survival of human pancreatic islets.
Nitric oxide (NO) has been proposed as a possible mediator of beta-cell damage in human IDDM. This hypothesis is based on in vitro studies with rodent pancreatic islets. In the present study we examined whether human beta-cells are affected by NO. In view of species differences in beta-cell sensitivity to damaging agents, rat islets were investigated in parallel. Isolated islets were exposed for 90 min to different concentrations of three chemically unrelated NO donors, SIN-1, GSNO or RBS. At the end of this incubation, human insulin release was mostly similar in control and NO-treated islets but, 48 h later, islet retrieval, islet DNA and insulin content, and glucose-induced insulin release were markedly lower in islets exposed to NO donors. Rat islets were already inhibited during the initial 90 min; 48 h later their loss in beta-cell function was similar to that in human islets. Nicotinamide or succinic acid monomethyl ester partially protected against SIN-1 induced islet cell loss, but not against the functional inhibition of human pancreatic islets. Exposure of human or rat islets to RBS was associated with significant DNA strand breakage, as judged by the comet assay (single cell gel electrophoresis) and by ultrastructural signs of cell damage. DNA damage was more severe in rat islet cells exposed to similar amounts of RBS. It is concluded that NO donors can damage human pancreatic islets, an effect paralleled by induction of nuclear DNA strand breaks.
Adolescent↗
Protective effect of deoxyribonucleosides on UV-irradiated human peripheral blood T-lymphocytes: possibilities for the selective killing of either cycling or non-cycling cells.
Non-cycling human T-lymphocytes from normal subjects show a 10-fold greater sensitivity than fibroblasts to UV-B (280-315 nm) irradiation from a Westinghouse FS20 lamp, but only a 2.7-fold greater sensitivity to UV-C (254 nm) irradiation. Hypersensitivity is associated with a deficiency in the rejoining of excision breaks. Non-cycling T-lymphocytes have extremely low deoxyribonucleotide pools. Addition to the medium of the four deoxyribonucleosides, each at a concentration of 10(-5) M, substantially increases survival and reduces the persistence of excision-related strand breaks following UV-B or UV-C irradiation (Yew and Johnson (1979) Biochim. Biophys. Acta 562, 240-241; Green et al. (1994) Mutation Res., 315, 25-32). UV-resistance of T-lymphocytes is also increased by stimulating the cells into cycle. The addition of deoxyribonucleosides does not further enhance survival of cycling cells and they do not reach the level of resistance achieved by non-cycling cells in the presence of deoxyribonucleosides. We suggest that two opposing effects are in operation. Cells out of cycle can show increased resistance to DNA damage in the absence of division but they also have reduced deoxyribonucleotide pools, which may limit DNA repair. With UV-B irradiation, the exceptionally low dNTP pools in non-cycling T-lymphocytes cause this second effect to predominate. In contrast, with ionising radiation, which forms highly cytotoxic double-strand breaks, non-cycling human T-lymphocytes are slightly more resistant than fibroblasts. Non-cycling cells such as T-lymphocytes should be especially sensitive to agents which produce a high proportion of read excisable damage, but should show normal resistance to agents which highly toxic lesions. It may be possible by choice of DNA damaging agent and manipulation of cellular deoxyribonucleotide pools, to choose regimes which will selectively kill either cycling or non-cycling cells and to improve the efficacy of standard therapeutic procedures. Conditions favouring selective killing of non-dividing T-lymphocytes but sparing stem cells may be of value in bone marrow transplantation. Conditions favouring selective killing of dividing cancer cells but sparing non-dividing normal tissue may be of value in cancer therapy.
Cell Cycle↗
Comet assay to detect nitric oxide-dependent DNA damage in mammalian cells.
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Animals↗
Quantitative and conceptual contributions of mathematical modeling to current views on vitamin A metabolism, biochemistry, and nutrition.
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Animals↗
Tumor necrosis factor-alpha and interferon-gamma inhibit insulin secretion and cause DNA damage in unweaned-rat islets. Extent of nitric oxide involvement.
Nitric oxide has been implicated as one possible mediator of interleukin-1 beta (IL-1)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5-500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14C-labeled arginine to 14C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with NG-monomethyl-L-arginine, resulted in inhibition of nitrite formation by 5-1,000 U/ml IFN+TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF+IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF+IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitric-oxide-mediated events predominate.
Animals↗
Lack of evidence for an association between the frequency of mutants or translocations in circulating lymphocytes and exposure to radon gas in the home.
Radon measurements in the living room and main bedroom of 41 houses in the town of Street, Somerset, England have been made. Exposure levels, weighted using the formula of the UK National Radiological Protection Board, of 19-484 Bq m-3 (about half > 100 Bq m-3) were found. Blood samples were obtained from a total of 66 occupants in these homes, and the frequency of genetic alterations in lymphocytes was estimated using two different end points. Gene mutations at the hypoxanthine guanine phosphoribosyl transferase locus were determined in T lymphocytes for 65 subjects using a clonal assay, and the frequency of the BCL-2 t(14;18) translocation, a chromosomal event associated with leukemia/lymphoma, was estimated in lymphocytes using a polymerase chain reaction-based technique for 64 subjects. In neither case was a significant correlation with radon levels in the home found, in contrast to our earlier observation with a smaller series.
Adult↗
Comparison of inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans by streptozotocin and methyl and ethyl nitrosoureas and methanesulphonates. Lack of correlation with nitric oxide-releasing or O6-alkylating ability.
We have studied inhibition of glucose-stimulated insulin secretion in islets of Langerhans isolated from adult Sprague-Dawley rats and treated with different alkylating agents. Streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU) all released nitric oxide, as demonstrated by an increase in medium nitrite and cellular cyclic GMP. Methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), which do not possess a nitroso group, did not show evidence of nitric oxide release. All five compounds, however, decreased glucose-stimulated insulin release, suggesting that nitric oxide release was not necessary for the inhibition of secretion. Lack of involvement of nitric oxide was further suggested by the failure of oxyhaemoglobin to reverse STZ and MNU inhibition of insulin secretion. Since ENU was at least as effective as MNU in inhibiting insulin secretion, it appears that alkylation of DNA at the O6 position of guanine may not be involved in this process.
Alkylating Agents↗
Induction of cyclobutane pyrimidine dimers, pyrimidine(6-4)pyrimidone photoproducts, and Dewar valence isomers by natural sunlight in normal human mononuclear cells.
Immunocytochemistry was used for the direct measurement of cyclobutane pyrimidine dimers, (6-4) photoproducts, and Dewar isomers in normal human mononuclear cells following irradiation by natural sunlight or by a FS20 broad spectrum UVB sunlamp. The induction of each type of photoproduct was detected following 30-60 min sunlight exposure or with FS20 fluences as low as 50-100 Jm-2. With increasing FS20 fluences, there was a dose-dependent increase in the binding of pyrimidine dimer, (6-4) photoproduct, and Dewar isomer-specific monoclonal antibodies. The relative ratio of Dewar isomer to (6-4) photoproduct antibody binding sites was much higher following exposure to natural sunlight than to broad spectrum UVB. With the (6-4) monoclonal antibody, a small increase in binding sites was evident after a 1-h exposure to natural sunlight. This remained relatively constant with further exposure. These results are consistent with the hypothesis that, following irradiation with natural sunlight, the majority of (6-4) photoproducts are converted into Dewar valence isomers.
Antibodies, Monoclonal↗
Compartmental analysis of norepinephrine kinetics in congestive heart failure.
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Adult↗
Synergistic effect of growth factors on cell outgrowth from explants of rabbit anterior cruciate and medial collateral ligaments.
Cellular migration and proliferation are integral aspects of wound healing. An in vitro assay for cellular migration and proliferation using explants of rabbit anterior cruciate and medial collateral ligaments was developed previously. This study presents the effects of serum-free culture medium supplemented with basic fibroblast growth factor, bovine insulin, transforming growth factor-beta 1, and platelet-derived growth factor-B, added either individually or in combination, on cell outgrowth in explants of rabbit anterior cruciate and medial collateral ligaments. Outgrowth was assessed at 3 and 6 days by counting the number of cells surrounding the tissue explants. For explants of both ligaments, cell outgrowth was dependent on the presence of 10% fetal bovine serum or the combination of growth factors. Little outgrowth occurred in explants of either ligament in the presence of basic fibroblast growth factor, transforming growth factor-beta 1, or bovine insulin. Platelet-derived growth factor-B at concentrations of 20 and 100 ng/ml seemed to increase cell outgrowth from medial collateral ligament explants at 6 days. The outgrowth from the explants of both ligaments was much greater in the presence of all four growth factors than the sum of the outgrowth with the individual factors. This synergistic effect was as much as 10-fold and 20-fold for the anterior cruciate ligament explants at days 3 and 6, respectively, but no more than 3-fold for the medial collateral ligament explants at these times. Medial collateral ligament explants exhibited greater cell outgrowth than anterior cruciate ligament explants in 10% serum and in the presence of the four growth factors.
Animals↗
Comparison of effects of iron and calcium chelators on the response of L5178Y sublines to X-rays and H2O2.
The L5178Y murine lymphoma subline LY-R is twofold more resistant to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by hydrogen peroxide: at 37 degrees C LY-R cells are 1.4 times more sensitive to the killing effect of H2O2 than LY-S cells. Pretreatment with the iron chelator desferroxamine followed by hydrogen peroxide treatment at 37 degrees C gives a considerable sparing effect, which is substantially greater for the LY-R subline than for the LY-S subline. This is reflected in the initial DNA damage (estimated by single cell gel electrophoresis), survival and mutation frequency in the HGPRT locus. Similar results have been obtained with calcium chelators, which, according to recent findings (Sandström and Granström 1993), also are efficient iron chelators. In contrast, the response to X-rays is not modified by the above chelators, with the exception of mutation frequencies: lower mutant numbers are found in desferroxamine pretreated LY-R cells.
Aminoquinolines↗
Suggestions concerning the relationship between mutant frequency and mutation rate at the hprt locus in human peripheral T-lymphocytes.
Mutant frequency is defined as the proportion of mutant cells in a population and is readily estimated. It should be distinguished from mutation rate, which relates to the rate at which mutation events arise, and is generally expressed as events per cell division. Since one mutation event may give rise to one or many mutant cells, depending on the generation in which it has arisen, the relationship of mutant frequency to the underlying mutation rate is complex. A large number of estimates of mutant frequency at the hprt locus in human lymphocytes are available, from our two laboratories among others. From our two extensive data sets, we have determined median hprt mutant frequencies of different age groups and used the method of Lea and Coulson (J. Genet., 49, 1949, 264-285) to attempt to estimate the underlying mutation rate at this locus. It is in principle possible to obtain estimates of mutation rate from the mutant frequency in newborns, from the increase in mutant frequency with age, and from the difference between the upper and lower quartile mutant frequencies. We discuss reasons for the discrepancies between these estimates and argue that the best estimate can probably be obtained from the increase in mutant frequency with age. We arrive at an estimate of mutation rate to 6-thioguanine resistance at the hprt locus of about 5 x 10(-7) mutation events per nominal cell division.
Adolescent↗