Comet assay to detect nitric oxide-dependent DNA damage in mammalian cells.
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Animals↗
Biomedical subjects
J E Lowe
Publications and source records attributed to J E Lowe.
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↗
Predictors of outcome of tricuspid valve replacement in carcinoid heart disease.
The cardiac valvular surgical experience of patients in the Duke Carcinoid Database was reviewed to assess operative outcome. Of the 604 patients in the database, 19 patients with carcinoid heart disease were identified by cardiac catheterization or echocardiography, or both. Eight of these underwent tricuspid valve replacement surgery with bioprostheses (2 also had open pulmonic valvuloplasty). Compared with patients medically managed, surgically treated patients were similar with the exception that they had higher right atrial mean (17 +/- 6 vs 9 +/- 4 mm Hg, p = 0.03) and v-wave (27 +/- 6 vs 17 +/- 7 mm Hg, p = 0.04) pressures. Of the 8 surgical patients, 5 (63%) died within 30 days. Causes of death included tricuspid valve thrombosis, cerebral vascular accident, coagulopathy, renal failure, and intractable right heart failure. High comorbidity was present in all 8 patients. There was a weak trend (p = 0.17) toward lower Charlson comorbidity indexes in survivors (6.7 +/- 0.6) compared with nonsurvivors (7.6 +/- 0.9). Age was significantly lower (p = 0.036) in survivors (46 +/- 13 years) compared with nonsurvivors (69 +/- 4 years). Extended follow-up revealed 2 patients who survived beyond a decade. Review of 47 carcinoid valve replacement cases (Duke Carcinoid Database and 39 published cases) revealed a 30-day mortality of 56% for patients > 60 years of age, and 0% for those < or = 60 years of age (p < 0.0001). Although valve replacement surgery can afford prolonged palliation from carcinoid heart disease, it is associated with a significant mortality risk. Careful preoperative risk stratification by age and comorbidity may provide a means for optimal selection of surgical candidates.
Adult↗
Stimulation of non-oxidative glucose utilization by L-carnitine in isolated myocytes.
The effects of L-carnitine on 14CO2 release from [1-14C]pyruvate oxidation (an index of pyruvate dehydrogenase activity, PDH), [2-14C]pyruvate, and [6-14C]glucose oxidation (indices of the acetyl-CoA flux through citric acid cycle), and [U-14C]glucose (an index of both PDH activity and the flux of acetyl-CoA through the citric acid cycle), were studied using isolated rat cardiac myocytes. L-carnitine increased the release of 14CO2 from [1-14C]pyruvate, and decreased that of [2-14C]pyruvate in a time and concentration-dependent manner. At a concentration of 2.5 mM, L-carnitine produced a 50% increase of CO2 release from [1-14C]pyruvate and a 50% decrease from [2-14C]pyruvate oxidation. L-carnitine also increased CO2 release from [1-14C]pyruvate oxidation by 35%, and decreased that of [2-14C]pyruvate oxidation 30%, in isolated rat heart mitochondria. The fatty acid oxidation inhibitor, etomoxir, stimulated the release of CO2 from both [1-14]pyruvate and [2-14C]pyruvate. These results were supported by the effects of L-carnitine on the CO2 release from [6-14C]- and [U-14C]glucose oxidation. L-carnitine (5 mM) decreased the CO2 release from [6-14C]glucose by 37%, while etomoxir (50 microM) increased its release by 24%. L-carnitine had no effect on the oxidation of [U-14C]glucose. L-carnitine increased palmitate oxidation in a time- and concentration-dependent manner in myocytes. Also, it increased the rate of efflux of acetylcarnitine generated from pyruvate in myocytes. These results suggest that L-carnitine stimulates pyruvate dehydrogenase complex activity and enhances non-oxidative glucose metabolism by increasing the mitochondrial acetylcarnitine efflux in the absence of exogenous fatty acids.
Acetylcarnitine↗
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↗
In-hospital and long-term outcome after porcine tricuspid valve replacement.
Porcine bioprostheses are often used for tricuspid valve replacement, yet the long-term outcome after this procedure is not well documented. Therefore, the records of 129 patients undergoing tricuspid valve replacement with Carpentier-Edwards (n = 88) or Hancock (n = 41) prostheses between 1975 and 1993 were reviewed. The operation required a repeat median sternotomy in 66 of 129 (51%) patients, whereas 67 of 129 (52%) underwent double or triple valve replacement. Operative mortality was 14% (2/14) in patients undergoing first-time isolated tricuspid valve replacement and 27% (35/129) overall. Survival at 5, 10, and 14 years was 56% +/- 5%, 48% +/- 5%, and 31% +/- 9%, and freedom from tricuspid reoperation at 5, 10, and 14 years was 96% +/- 3%, 93% +/- 4%, and 49% +/- 17%. No valve thrombosis was observed. In this largest reported series of porcine bioprostheses in the tricuspid position, long-term freedom from valve-related events was excellent because of a low incidence of valve thrombosis and a valve durability of 13 to 15 years in a population with limited life expectancy.
Bioprosthesis↗
Hyperinsulinemia inhibits hepatic peroxisomal beta-oxidation in rats.
Studies show that insulin deficiency enhances peroxisomal enzyme activities. It is not known, however, whether hyperinsulinemia exerts the opposite effect on peroxisomes. Male Sprague-Dawley rats were infused with normal saline, glucose or galactose for 7 days. Only glucose caused an increase in serum insulin levels. The increase in insulin secretion, in response to glucose, was blocked with diazoxide. Data show an inverse relationship between serum insulin levels and hepatic peroxisomal beta-oxidation (r2 = 0.90, p < 0.01). While hyperinsulinemic rats had diminished peroxisomal beta-oxidation, lowering serum insulin restored peroxisomal enzyme activity to normal levels. These effects were independent of blood glucose levels (r2 = 0.35). In addition to decreasing peroxisomal beta-oxidation, hyperinsulinemia was accompanied by accelerated animal mortality, an effect which was also prevented by lowering serum insulin levels. Peroxisomal deficit may be a potentially lethal consequence of hyperinsulinemia.
Animals↗
Reduced induction of carbohydrate utilization by inhibition of fatty acid oxidation in myocytes from diabetic animals.
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Animals↗
Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.
By using specific monoclonal antibodies in situ and a computer-assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6-4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad-spectrum UVB and narrow-spectrum UVB. The lamps produced these lesions in different proportions, with broad-spectrum UVB inducing a greater combined yield of (6-4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow-spectrum UVB. The relative induction ratios of (6-4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad- or narrow-spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad-spectrum or narrow-spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T-lymphocytes and fibroblasts at fluences, which induce equivalent yields of cyclobutane dimers, (6-4) photoproducts or (6-4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6-4) photoproduct formation, whereas killing of T-lymphocytes seems to be mediated by combined (6-4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.
Antibodies, Monoclonal↗
DNA strand breakage, cytotoxicity and mutagenicity of hydrogen peroxide treatment at 4 degrees C and 37 degrees C in L5178Y sublines.
Cells from the L5178Y murine lymphoma subline LY-R are 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. Cells of the two sublines in logarithmic growth phase were treated with hydrogen peroxide in phosphate-buffered saline for 1 h at 4 degrees C or 37 degrees C. From the comparison of D(o) values it followed that at 37 degrees C LY-R were 3.6 times more sensitive to the killing effect of H2O2 than LY-S cells; at 4 degrees C they were 11 times more sensitive. Treatment with hydrogen peroxide at 4 degrees C gave a considerable sparing effect, which was substantially greater for the LY-S subline; for LY-S cells D(o) was 5.7 times lower at 37 degrees C than at 4 degrees C, for LY-R cells only 1.9 times. The mutation frequency (HGPRT) in LY-R cells was increased in proportion to H2O2 concentration and was the same at both treatment temperatures. In contrast, mutation frequencies initially increased, then decreased with increasing H2O2 concentration in LY-S cells treated at 4 or 37 degrees C. The concentration at which the decline was initiated was higher at 4 than at 37 degrees C. DNA damage after H2O2 treatment (both temperatures, 5 min) was estimated from the 'comet' assay (single-cell gel electrophoresis). The initial damage, but not the residual damage, differed significantly in LY sublines. A period of slower repair (between 3 and 10 min) was found in LY-R cells.
Animals↗
Pulmonary mucormycosis: results of medical and surgical therapy.
Mucormycosis is an opportunistic fungal infection that commonly begins by invading the respiratory tract. The purpose of the present study was to define the clinical presentation of pulmonary mucormycosis and to evaluate current treatment regimens. Thirty patients treated at our institution and 225 cases reported in the literature were reviewed. For the combined groups, the mean age at presentation was 41 +/- 21 years and associated medical conditions included leukemia or lymphoma (37%), diabetes mellitus (32%), chronic renal failure (18%), history of organ transplantation (7.6%), or a known solid tumor (5.6%). The in-hospital mortality was 65% for patients with isolated pulmonary mucormycosis, 96% for those with disseminated disease, and 80% overall. The mortality in patients treated surgically was 11%, significantly lower than the 68% mortality in those treated medically (p = 0.0004). The most common causes of death were fungal sepsis (42%), respiratory insufficiency (27%), and hemoptysis (13%). Pulmonary mucormycosis has a high mortality; however, antifungal agents appear to improve survival. In addition, surgical resection may provide additional benefit to patients with pulmonary mucormycosis confined to one lung.
Adolescent↗
Right thoracotomy for reoperative right coronary artery bypass procedures.
The use of a right thoracotomy is a safe alternative approach for reentering the mediastinum for reoperative right coronary artery bypass grafting. This technique was used in 9 patients and allowed minimal dissection, reducing the risks of hemorrhage and injury to previously placed patent grafts. Cardiopulmonary bypass was used in 4 patients in whom myocardial protection was accomplished with systemic hypothermia. Right atrial cannulation provided adequate venous return, with arterial inflow established via the femoral artery (n = 2) or ascending aorta (n = 2). In 5 patients, revascularization was accomplished by temporary coronary artery occlusion without cardiopulmonary support. There were no deaths, and postoperative recovery was not delayed. Postoperative chest drainage (mean +/- standard deviation, 1,076 +/- 718 mL) was significantly less (p < 0.01) than redo median sternotomy (1,352 +/- 602 mL) in a similar population. Eight of 9 patients remain free of angina 29 +/- 24.8 months postoperatively.
Aged↗
Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells.
We have measured DNA strand breaks induced by ionising radiation in nucleated cells from freshly isolated whole blood from normal human subjects. Samples were taken after subjects had fasted overnight and again 1 h after they had eaten breakfast in combination with approximately 35 mg/kg vitamin C. Damage was measured by single cell gel electrophoresis (the 'comet' assay), in which DNA single strand breaks generate a comet tail streaming from the nucleus. In repeat experiments on 6 subjects a reduction in DNA damage, as indicated by a highly significant decrease in overall comet length, was observed following vitamin C ingestion, both in the unirradiated control blood samples and in the dose response to ionising radiation damage. In addition, consistent differences in dose response between individual subjects were found. The peak effect was 4 h after intake of food and vitamin C. An effect was also seen with vitamin C alone and after breakfast without additional vitamin C. Protection against strand breakage was also seen in Ficoll-separated mononuclear cells but evidence was not obtained for protection of separated, mitogen stimulated T-lymphocytes either against ionising radiation cell killing in a clonal assay, or against clastogenicity assessed by micronucleus formation following one cell division. Exposure of separated lymphocytes in vitro to vitamin C, at doses greater than 200 microM, did not offer protection but induced strand breakage. Our results raise the possibility that variation in normal diet may not only affect susceptibility to endogenous oxidative damage, but may affect some responses of the individual to radiation.
Artifacts↗
Effect of deoxyribonucleosides on the hypersensitivity of human peripheral blood lymphocytes to UV-B and UV-C irradiation.
We have previously shown that non-cycling (unstimulated) human lymphocytes from normal donors show extreme hypersensitivity to UV-B irradiation, and are killed by an excisable lesion which is not a pyrimidine dimer or 6-4 photoproduct. In this paper we show that addition of the 4 deoxyribonucleosides to the medium, each at 10(-5) M, substantially increased the survival of non-cycling normal human T-lymphocytes following UV-B irradiation and substantially reduced the frequency of excision-related strand breaks in human mononuclear cells. Addition of ribonucleosides to the medium did not enhance excision-break rejoining. The survival of fibroblasts, of cycling T-lymphocytes and of unstimulated xeroderma pigmentosum T-lymphocytes was not enhanced by deoxyribonucleosides. This suggests that the hypersensitivity is due to reduced rejoining of excision breaks as a consequence of low intracellular deoxyribonucleotide pools and that it can be redressed by supplementation of the medium with deoxyribonucleosides or upregulation of ribonucleotide reductase following mitogen stimulation. We suggest that UV-B forms an additional DNA lesion which is not a pyrimidine dimer or 6-4 photoproduct, which is relatively common, and at which incision is particularly efficient. In fibroblasts, repair of this lesion is completed with high efficiency, whereas in normal unstimulated T-lymphocytes, rapid incision exacerbates the effects of the reduced rate of strand rejoining and leads to cell death.
Cells, Cultured↗
Regulation of glucose utilization during the inhibition of fatty acid oxidation in rat myocytes.
The study of the regulation of glucose utilization by inhibition of fatty acid oxidation is greatly enhanced by the availability of specific inhibitors of fatty acid oxidation. This study examines the regulation of cardiac glucose utilization by inhibition of fatty acid oxidation at different sites. The effects of Etomoxir and 4-bromocrotonic acid (4-BCA) on the oxidation of [1-14C]palmitate, [1-14C]-octanoate and [U-14C]glucose were studied in isolated rat myocytes. Fifty percent inhibition of palmitate oxidation was achieved at 8 microM Etomoxir and 40 microM 4-BCA. Octanoate oxidation was inhibited only by 4-BCA. In contrast to their effect on palmitate oxidation, these inhibitors significantly stimulated the oxidation of glucose in a concentration-dependent manner. Moreover, the oxidation of [2-14C]pyruvate was increased two-fold by these compounds. The rate of utilization of [U-14C]-2-deoxyglucose was also stimulated 2-3 times by these inhibitors. These studies suggest that the stimulation of glucose utilization via the inhibition of fatty acid oxidation may be mediated through the stimulation of both glucose transport and the oxidation of pyruvate by the pyruvate dehydrogenase complex.
Animals↗
Thoracic intrathymic thyroid.
OBJECTIVE: The authors introduce thoracic intrathymic thyroid as a clinical entity. SUMMARY BACKGROUND DATA: Although accessory aberrant thyroid has not been found in other tissues in the mediastinum, a thoracic intrathymic location has not been described previously. It is believed that mediastinal thyroid tissue represents accessory ectopic tissue from the median thyroid anlage. Moreover, the close association of the thymus and thyroid supports the theory that mediastinal ectopic thyroid tissue develops from abnormal descent of these structures during embryogenesis. METHODS: Benign thoracic intrathymic thyroid lesions are described in patients with mediastinal masses. CONCLUSION: Thoracic intrathymic thyroid is a distinct entity. Its occurrence is supported both clinically and embryologically.
Adult↗
Comparison of anticoagulation regimens after Carpentier-Edwards aortic or mitral valve replacement.
BACKGROUND: To identify the optimal use of anticoagulants after Carpentier-Edwards valve replacement, a retrospective study of all patients undergoing Carpentier-Edwards aortic (N = 378) or mitral (N = 370) valve replacement was done. METHODS AND RESULTS: At the time of hospital discharge, 103 patients were managed with warfarin, 509 with aspirin alone, and 136 with no anticoagulation or antiplatelet therapy. Over the first 90 days after aortic or mitral valve replacement, the linearized rate of hemorrhage was greater for warfarin than for aspirin or no therapy (16.7 +/- 7.6%, 3.4 +/- 1.7%, and 3.1 +/- 3.1% per patient-year, respectively; P = .03). After aortic valve replacement, aspirin provided a low rate of thromboembolism (0.8 +/- 0.2% per patient-year), not significantly different from warfarin or no treatment (2.9 +/- 1.6% and 1.5 +/- 0.6% per patient-year) (P = .07). After mitral valve replacement, no single treatment was most advantageous because the rate of hemorrhage over the first 90 days for warfarin was equivalent to the 90-day rate of thromboembolism with aspirin or no therapy. CONCLUSIONS: Anticoagulation after Carpentier-Edwards mitral valve replacement may be best guided by individual patient characteristics. Within the limits of a retrospective analysis, these data support the routine use of aspirin alone after Carpentier-Edwards aortic valve replacement, both in the first 90 days and long-term.
Aortic Valve↗
Measurements of blood flow and xanthine oxidase activity during postischemic reperfusion of the large colon of ponies.
To assess right colic artery blood flow and relevance of xanthine dehydrogenase/xanthine oxidase after experimentally induced strangulation obstruction and reperfusion of the colon, 5 ponies were subjected to 2.5 hours of complete ischemia of the left dorsal and ventral colons, allowed to recover from surgery, and monitored during a 48-hour reperfusion period. Five ponies were subjected to sham surgery and served as controls. All ponies had a Doppler ultrasound blood flow monitor implanted on the right colic artery near the pelvic flexure 10 to 14 days prior to the ischemic period. Colic artery blood flow was monitored prior to, during, and for 4 hours after surgery. Blood samples from the right colic artery and vein distal to the obstruction site were collected during surgery (prior to ischemia, after 1 and 2 hours of ischemia, and after 10 and 60 minutes of reperfusion) for determination of arterial and venous blood gas tensions and electrolytes. Prior to surgery, blood selenium and plasma vitamin E (alpha-tocopherol) concentrations and blood glutathione peroxidase (GPX) activity were determined to assess the status of endogenous antioxidants. Combined xanthine dehydrogenase (XDH) plus xanthine oxidase (XO) activity, and XO activity alone (nanomoles per minute per gram of tissue) were determined, using a dual-spectrophotometric technique. Xanthine dehydrogenase and oxidase activities were determined prior to ischemia, after 1 and 2 hours of ischemia, and at 1 and 48 hours after reperfusion. Median blood flow in the experimental and control groups (156 ml/min and 110 ml/min, respectively) was not statistically different before surgery, and was significantly (P < 0.02) lower in the experimental (4 ml/min) vs the control group (72.5 ml/min) during the ischemic period. Experimental ponies had significantly (P < 0.03) lower right colic artery blood flow during the 4 hours immediately after recovery from anesthesia. Significant difference was not observed in right colonic venous bicarbonate concentration between groups at any time. Median right colonic venous PCO2, pH, and standard base excess were different (P < 0.001) between groups during the ischemic period only. Median venous oxygen saturation and median venous PO2 were significantly (P < 0.001) lower in the experimental ponies at the end of 2 hours of ischemia, but were significantly (P < 0.05) increased during the reperfusion phase. Median venous potassium concentration was significantly (P < 0.01) higher in experimental ponies during the ischemic and reperfusion phases. Vitamin E and GPX values were within normal limits for all ponies.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗