Morphological characterization of small hepatocytes after bile duct ligation in chicken.

Small hepatocytes have hepatocyte-like characteristics and high proliferation activity. Most small hepatocyte studies report on in vitro rat or human hepatocytes. Only a few studies of small hepatocytes after bile duct ligation have been reported, and none of these have focused on these cells in birds. In this study, small hepatocytes appearing in bile duct ligation chicken liver were examined using the morphological method with histological, immunohistochemical, and ultrastructural studies. Nine Boris Brown hens (over 744-d old) were used. In all chickens, both the common hepatoenteric duct and hepatocystic duct were ligated, and the livers were examined 1, 4, 6, 9, and 13 weeks after bile duct ligation. Histologically, the small cells were half the size of normal liver cells, and mitotic figures were often observed. The nuclei showed two forms: large and small. Many small cells were negative for periodic acid-Schiff stain, but positive cells were rarely observed. The cells existed in colonies on the side of the sinusoid of the hepatic lamina. Immunohistochemically, the small cells with large nuclei were strongly positive for CD44, albumin and proliferation cell nuclear antigen, and the cells with small nuclei were weakly positive. In the CD44-positive cell colony, negative cells were often observed to have mature hepatocyte-like morphology. Moreover, many of the cells were PAN cytokeratin negative. Ultrastructurally, the small cells had more nuclei with rich heterochromatin, poor cytoplasmic organelles, and narrow cytoplasm with a high electron density than mature hepatocytes. Moreover, cells having a middle ultrastructural characteristic existed between the small cells and mature hepatocytes. The small hepatocellular colony of the chicken appeared as a regeneration-related change in the liver after bile duct ligation. The cell had high cell proliferation activity and morphological, immunohistochemical, and ultrastructural characteristics similar to those of the mammalian small hepatocyte, as well as a similar progenitor cell.

Preliminary analysis for integration of spot-scanning proton beam therapy and real-time imaging and gating.

PURPOSE: Spot-scanning proton beam therapy (PBT) can create good dose distribution for static targets. However, there exists larger uncertainty for tumors that move due to respiration, bowel gas or other internal circumstances within the patients. We have developed a real-time tumor-tracking radiation therapy (RTRT) system that uses an X-ray linear accelerator gated to the motion of internal fiducial markers introduced in the late 1990s. Relying on more than 10 years of clinical experience and big log data, we established a real-time image gated proton beam therapy system dedicated to spot scanning. MATERIALS AND METHODS: Using log data and clinical outcomes derived from the clinical usage of the RTRT system since 1999, we have established a library to be used for in-house simulation for tumor targeting and evaluation. Factors considered to be the dominant causes of the interplay effects related to the spot scanning dedicated proton therapy system are listed and discussed. RESULTS/CONCLUSIONS: Total facility design, synchrotron operation cycle, and gating windows were listed as the important factors causing the interplay effects contributing to the irradiation time and motion-induced dose error. Fiducial markers that we have developed and used for the RTRT in X-ray therapy were suggested to have the capacity to improve dose distribution. Accumulated internal motion data in the RTRT system enable us to improve the operation and function of a Spot-scanning proton beam therapy (SSPT) system. A real-time-image gated SSPT system can increase accuracy for treating moving tumors. The system will start clinical service in early 2014.

Ultraviolet B irradiation of the mouse eye induces pigmentation of the skin more strongly than does stress loading, by increasing the levels of prohormone convertase 2 and α-melanocyte-stimulating hormone.

BACKGROUND: In previous studies, we made the unexpected finding that in mice, ultraviolet (UV)B irradiation of the eye increased the concentration of α-melanocyte-stimulating hormone (α-MSH) in plasma, and systemically stimulated epidermal melanocytes. AIMS: To compare the extent of the pigmentation induced by social and restraint stress (which activate the hippocampus-pituitary system) with that induced by UVB irradiation. METHODS: DBA/2 and sham-operated or hypophysectomized DBA/2 mice were subjected to local UVB exposure using a sunlamp directed at the eye, and two types of stress (social and restraint) were imposed. RESULTS: UVB irradiation of the eye or exposure to stress loading both increased the number of Dopa-positive melanocytes in the epidermis, and hypophysectomy strongly inhibited the UVB-induced and stress-induced stimulation of melanocytes. Irradiation of the eye caused a much greater increase in dopamine than did the stress load. Both UVB eye irradiation and stress increased the blood levels of α-MSH and adrenocorticotropic hormone (ACTH). In addition, the increase in plasma α-MSH was greater in animals subjected to UVB eye irradiation than in those subjected to stress loading, whereas the reverse occurred for plasma ACTH. UVB irradiation to the eye and stress loading increased the expression of prohormone convertase (PC)1/3 and PC2 in the pituitary gland. The increase in expression of pituitary PC2 was greater in animals subjected to UVB eye irradiation than to stress, whereas no difference was seen between the two groups for the increase in PC1/3. CONCLUSIONS: UVB eye irradiation exerts a stronger effect on pigmentation than stress loading, and is related to increased levels of α-MSH and PC2.

Ultraviolet B radiation to the eye induces pigmentation in the epidermis via the activation of the subunit gp91 phox of reduced nicotinamide adenine dinucleotide phosphate oxidase.

Irradiation by ultraviolet (UV)B is known to increase the number of dopamine (Dopa)-positive melanocytes in the skin. In this study, a 2.5-kJ/m(2) dose of UVB radiation was delivered by a sunlamp to the ear or the eye of wild-type C57BL/6j mice and of gp91 phox(-/-) C57BL/6j mice that had a knockout mutation of the gp91 phox subunit of reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH). The degree of change in the Dopa-positive melanocyte expression in was reduced in gp91 phox(-/-) mice given UVB irradiation to the eye, but not in those given irradiation to the ear. The plasma level of α-melanocyte-stimulating hormone (α-MSH) in the blood increased in the C57BL/6j mice after irradiation to either the eye or the ear, but it did not increase in the gp91 phox(-/-) mice given UVB irradiation to the eye. Both gp91 phox and α-MSH in the central nervous system seem to contribute to pigmentation after UVB irradiation of the eye in mice.

The α-melanocyte-stimulating hormone-melanocortin receptor system influences the effects of ultraviolet A on skin and intestinal immunity in mice.

Irradiation by ultraviolet (UV) A is known to decrease Langerhans cells (LCs) in the skin and increase IgA expression in the intestine, specifically the jejunum. These changes were induced in C57BL/6j mice by exposure of the ear or the eyes to 11 J/cm2 UVA radiation, then a melanocortin receptor agonist (Agouti-related protein; AgRP) was introduced either intracerebrally or intracerebroventricularly. The degree of change in both LC number and IgA expression induced by UVA eye irradiation was reduced more by intracerebral than by intraperitoneal injection of AgRP. α-Melanocyte-stimulating hormone and melanocortin receptors in the brain seem to contribute to immunomodulation after UVA irradiation of the eye or the ear in mice.

Increase in dopa-positive melanocytes in the mouse intestine in response to ultraviolet B rays via the eyes.

BACKGROUND: Irradiation by ultraviolet (UV) initiates pigmentation of skin; however, it is not known whether changes in intestinal pigmentation are also induced by UVB irradiation of the eye. AIM: To examine the influence of UVB irradiation of the eye or ear on the pigmentation of mouse epidermis and intestine. METHODS: DBA/2 male mice were locally exposed to UVB (280-320 nm) using a 20SE sunlamp directed at the eye or ear. The irradiation was given over 3 days, at a dosage of 2.5 kJ/m(2) per day. Five days after irradiation, samples were taken from the skin and intestine. Melanocytes in both epidermis and intestine were stained for dopa and expression of melanocortin-1 receptor (MC1R). Levels of plasma α-melanocyte-stimulating hormone (α-MSH) were measured using ELISA. RESULTS: Ultraviolet B irradiation of either the eye or ear in increased the number of dopa-positive melanocytes in the skin and the intestine (jejunum and colon). Irradiation of the eye caused a much greater increase in dopa than did irradiation of the ear. Both eye and ear irradiation increased blood α-MSH level to a similar extent, but only irradiation to the eye increased MC1R expression in the intestine. CONCLUSIONS: These results suggest that the UVB-induced pigmentation in the epidermis and the intestine is related to increased levels of α-MSH and MC1R.

Radical scavenging activity of bisbenzylisoquinoline alkaloids and traditional prophylactics against chemotherapy-induced oral mucositis.

BACKGROUND AND OBJECTIVE: Oral mucositis is a major severe toxic side-effect of systemic chemotherapy and irradiation in patients with cancer. Various free radical scavengers have been shown to prevent chemotherapy-induced skin necrosis. The objective of this study was to determine the antioxidant activity of a bisbenzylisoquinoline alkaloidal compound (BIQAC) and a series of chemicals, including allopurinol, used clinically for the treatment of chemotherapy-induced mucositis. METHODS: Allopurinol, melatonin, camostat mesilate, gabexate mesilate, hydroquinone and BIQAC were tested for their radical scavenging activities on four different radical species: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) cation radical (ABTS(*+)) using standard methods, and superoxide anion radical (O(2) (-)) and hydroxyl radical (OH(*)) using electron spin resonance. RESULTS: Allopurinol had radical scavenging activity against O(2) (-) only. Melatonin had strong radical scavenging activity against ABTS(*+), and weak activity against DPPH radical and OH(*). Camostat mesilate had weak radical scavenging activity against OH(*). Gabexate mesilate had no radical scavenging activity against any of these radicals. Hydroquinone had strong radical scavenging activity against DPPH radical and ABTS(*+), and moderate activity against both O(2) (-) and OH(*). BIQAC had moderate radical scavenging activity against DPPH radical, strong radical scavenging activity against ABTS(*+) and O(2) (-), and weak activity against OH(*). CONCLUSION: The BIQAC had the most braod-spectrum radical scavenging activity, suggesting that it may be effective against chemotherapy-induced mucositis. These findings also suggest that this radical-scavenging activity screening method, against four kinds of radicals, may be useful for the screening of radical scavenging activity of new natural and synthetic chemicals.

Variance components due to direct genetic, maternal genetic and permanent environmental effect for growth and feed-efficiency traits in young male Japanese Black cattle.

Variance components and genetic parameters were estimated using data recorded on 740 young male Japanese Black cattle during the period from 1971 to 2003. Traits studied were feed intake (FI), feed-conversion ratio (FCR), residual feed intake (RFI), average daily gain (ADG), metabolic body weight (MWT) at the mid-point of the test period and body weight (BWT) at the finish of the test (345 days). Data were analysed using three alternative animal models (direct, direct + maternal environmental, and direct + maternal genetic effects). Comparison of the log likelihood values has shown that the direct genetic effect was significant (p < 0.05) for all traits and that the maternal environmental effects were significant (p < 0.05) for MWT and BWT. The heritability estimates were 0.20 +/- 0.12 for FI, 0.14 +/- 0.10 for FCR, 0.33 +/- 0.14 for RFI, 0.19 +/- 0.12 for ADG, 0.30 +/- 0.14 for MWT and 0.30 +/- 0.13 for BWT. The maternal effects (maternal genetic and maternal environmental) were not important in feed-efficiency traits. The genetic correlation between RFI and ADG was stronger than the corresponding correlation between FCR and ADG. These results provide evidence that RFI should be included for genetic improvement in feed efficiency in Japanese Black breeding programmes.

Immunological cell situation in the skin of atopic model mice.

BACKGROUND: We observed nishikinezumi, cinnamon-coloured (NC)/Fujita (F) mice aged between 5 and 28 weeks. These NC mice have skin eruptions that resemble human atopic dermatitis (AD) under conventional circumstances. OBJECT: We investigated the skin of eruptive and non-eruptive lesions in NC/F mice by using haematoxylin-eosin (H&E) staining, toluidine blue staining and immunohistopathological study with immunoglobulin (Ig)EepsilonRI, CD23, interleukin (IL)-4, IL-5, interferon (INF)-gamma and Ia antigen. RESULTS: Histological examination of the eruptive lesions revealed the perivascular infiltration of many lymphocytes and mast cells into the upper dermis. Intracellular oedema of the epidermis, lymphocyte infiltration into the epidermis and liquefaction degeneration of the basal layer were also observed. The numbers of IL-4 and IL-5 positive cells in the eruptive lesions were larger than those of the non-eruptive lesions. IL-4 and IL-5 positive cells in the eruptive lesions increased weekly. Some IFN-gamma positive cells were observed in the eruptive lesions after 21 weeks. IFN-gamma positive cells were scarce in the skin of both the non-eruptive and eruptive lesions before 21 weeks. Serum IgE increased from 7 weeks to 21 weeks. DISCUSSION: We confirmed that these findings indicated that T helper (Th)2-dominant immunological activation transformed to a Th1-dominant situation. Many IgEepsilonRI positive cells were recognized in the dermis of the eruptive lesions by the time IgE had decreased. We assumed that the dermatitis before 21 weeks was an IgE-mediated allergy. We have previously reported that older NC/F mice had positive patch-test reactions to mites. Because serum IgE decreased after 21 weeks, dermatitis after 21 weeks might be associated more with cell-mediated delayed hypersensitivity than with IgE-mediated immediate allergy.

Oxidative stress promotes the regression of fetal liver hemopoiesis.

Although apoptosis is believed to play an important role in the ontogenetic development of animals, the molecular mechanism that triggers the regression of liver hemopoiesis during the perinatal period is not known. Apoptosis is induced by many factors such as a decrease in growth factors and increased oxygen stress. Since hepatic gamma-glutamyl transferase (GT) levels change markedly during the perinatal period in rodents, the metabolism of glutathione (GSH), a naturally occurring major antioxidant, might change significantly in and around liver cells. Hemopoietic cells but not hepatocytes exhibit significant apoptosis in thiol-free medium and the hemopoietic apoptosis can be inhibited by various thiols, such as L-cysteine, N-acetyl-L-cysteine, and GSH. The contribution of GSH levels in and around fetal liver cells in the triggering of apoptosis in hemopoietic cells is discussed.

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions.

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H2O2, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine-Fe(II), and N-methyl-D-glucaminedithiocarbamate (MGD)-Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine-Fe(II)-NO, and 2 cysteine-Fe(II)-2 NO complexes, respectively, and three line signals due to MGD-Fe(II)-NO were observed. Considerable amount of NO were liberated as determined by NO2-, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.

Standardization of nitric oxide aqueous solutions by modified Saltzman method.

Nitric oxide (NO) aqueous solutions were prepared by saturating pure NO gas and hydrolyzing 1 mM 1-hydroxy-2-oxo-3-(N-methyl-3-aminoethyl)-3-methyl-1-triazene (NOC-7), a NO donor, under anerobic conditions. The modified Saltzman method was employed for standardization of the NO aqueous solutions. NO and NO(2) in the solutions were driven with nitrogen gas stream into the first Saltzman solution to measure NO(2) and the leaked NO was driven with air stream through an oxidizing solution into the second Saltzman solution to measure NO, and NO(-)(2) and NO(-)(3) in the residual solutions were determined directly and after reduction with nitrate reductase, respectively. The concentrations of nitrogen oxide species in the NO solutions were about 1.8 mM NO/0.01 mM NO(2)/0.1 mM NO(-)(2)/0.1 mM NO(-)(3), and unchanged during keeping at 20 degrees C for 1 h under anerobic conditions but became 0.05 mM NO/0.01 mM NO(2)/1.7 mM NO(-)(2)/0.1 mM NO(-)(3) by keeping at 20 degrees C for 10 min under aerobic conditions. Instability of NO under aerobic conditions was supported by consumption of 1/4 equivalent amount of dissolved oxygen, and by loss of ability to convert 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) to carboxy-PTI. Simultaneous quantification of nitrogen oxide species by the modified Saltzman method was found to be useful for practical standardization of NO aqueous solutions.

Effect of food reductones, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and hydroxyhydroquinone (HHQ), on lipid peroxidation and type IV and I allergy responses of mouse.

The effect of long-term supplementation of food reductones, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) (2%, w/w), detected in many foodstuffs including soy sauce, and hydroxyhydroquinone (1,2,4-benzenetriol) (HHQ) (1.2%, w/w), detected in coffee, on mouse lipid peroxidation and type IV and I allergy responses was investigated. The effect of supplementation of these reductones combined with NO(2) inhalation (5-6 ppm) was also investigated. Levels of thiobarbituric acid-reactive substances in lung were remarkably increased, and those in kidney and liver were slightly decreased by supplementation of DMHF or HHQ. The degree of 2,4-dinitrochlorobenzene (DNCB)-sensitized lymph node cell proliferation as assessed by lymph node assay was remarkably enhanced by supplementation of DMHF or HHQ. Both the DNCB-sensitized and the trimellitic anhydride-sensitized increases in IgE levels of mice were enhanced to greater extent by supplementation of DMHF or HHQ. In no cases were additive effects of NO(2) inhalation observable. Allergen-sensitized type IV and I allergy responses of mice may be enhanced by supplementation of food reductones, DMHF or HHQ.

Release of nitric oxide together with carbon-centered radicals from N-nitrosamines by ultraviolet light irradiation.

Solutions of N-nitrosamines, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomorpholine and N-nitrosopyrrolidine in phosphate buffer (pH 7.4) were irradiated by ultraviolet (UV) light at room temperature. The N-nitrosamines were extensively degraded due to irradiation for 120 min in a time-dependent fashion as monitored by UV-absorption or high performance liquid chromatographic analysis. Carbon-centered radicals were generated from four N-nitrosamines during the short time irradiation of 10-60 s as monitored by electron spin resonance (ESR) technique using 5,5-dimethyl-1-pyrroline N-oxide and N-tert-butyl-alpha-phenylnitrone as spin traps. Nitric oxide (NO) was generated during the short time irradiation as monitored by ESR technique using cysteine-Fe(II) complex, N-methyl-D-glucamine dithiocarbamate and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Significant amounts of nitrite (4-16%) from four N-nitrosamines and also a significant amount of nitrate (4%) was produced from N-nitrosodimethylamine during the irradiation time of 120 min. Released NO from the N-nitrosamines must be converted into nitrite through intermediary reactive nitrogen oxide species including nitrogen dioxide and dinitrogen trioxide in contact with dissolved oxygen.

Total synthesis of reveromycin A.

The stereoselective total synthesis of reveromycin A (1), a potent inhibitor of eukaryotic cell growth, has been accomplished on the basis of the stereocontrolled construction of the 6,6-spiroketal system, efficient succinylation of the tert-alcohol under high pressure, and the introduction of the unsaturated side chains.

Is nitrotyrosine generated in human erythrocytes in circulation?

Nitrotyrosine is considered a stable biomarker of reactive nitrogen species, including nitrogen dioxide (NO2) and peroxynitrous acid (ONOOH) in biomaterials. There are inconsistent observations on the detection of free and protein-associated nitrotyrosine in normal human plasma. Human erythrocytes, differentiated from erythrocyte precursor cells in the bone marrow, circulating in the body for an average of 120 d, and finally removed by spleen macrophages, may be exposed to reactive nitrogen species. In the present study, membrane proteins and hemoglobin from the senescent erythrocyte population were compared with those from young erythrocytes separated from the same individuals in their nitrotyrosine presence using newly prepared rabbit polyclonal anti-nitrotyrosine-ribonuclease A and anti-nitro(N-butoxycarbonyl)tyrosine-bovine serum albumin antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the membranes and hemoglobin, and subsequent Western blot analysis, showed that these antibodies only slightly bind to the bands of the proteins from both young and senescent erythrocytes, whereas these antibodies definitely bind to the protein bands of membranes and hemoglobin nitrated by NO2 or ONOOH in vitro. This result indicates that nitrotyrosine is not detected in the membrane proteins and hemoglobin in human normal erythrocytes in circulation. However, this does not conclude that erythrocytes are not exposed to reactive nitrogen species in the circulation.

Effect of food reductones on the generation of the pyrazine cation radical and on the formation of the mutagens in the reaction of glucose, glycine and creatinine.

One of the possible pathways of the formation of mutagens in heated foods is through the pyrazine cation radical generated in the early stage of the Maillard reaction. The aim of the present study was to elucidate how food reductones contribute to the pyrazine cation radical generation in the reaction of glucose (Glc) and glycine (Gly), and to the formation of the mutagens in the reaction of Glc, Gly and creatinine. Electron spin resonance (ESR) studies showed that fragrant reductones, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), generated in the Maillard reactions, enhanced the generation of the pyrazine cation radical in the reaction of Glc and Gly, and the reaction of DMHF or HEMF with Gly generated a larger amount of the pyrazine cation radical than the reaction of Glc and Gly, indicating that the furanones were intermediates of the pyrazine cation radical. By contrast, food antioxidants, ascorbic acid and erythorbic acid, effectively scavenged the pyrazine cation radical generated in the reaction of Glc and Gly. DMHF and HEMF were not effective to modulate the mutagen formation in the reaction of Glc, Gly and creatinine, and the mutagenicity produced in the reaction of DMHF or HEMF, Gly and creatinine was lower than that produced in the reaction of Glc, Gly and creatinine. On the other hand, ascorbic acid and erythorbic acid were effective to decrease the mutagen formation in the reaction of Glc, Gly and creatinine.

Prevention of the formation of mutagenic and/or carcinogenic heterocyclic amines by food factors.

Introduction of phenolic antioxidants, thiol compounds and reductones into the heated model system composed of glucose/glycine/creatinine was effective to scavenge the intermediary pyrazine cation radical and to reduce the mutagenicity due to imidazoquinoxaline heterocyclic amines. Addition of a reductone, ascorbate or erythorbate, at 0.3% to ground beef effectively reduced the mutagenicity of cooked hamburger. Mutagenicity of cooked hamburger was also decreased by addition of glucose at more than 0.7% to ground beef.

Participation of the pyrazine cation radical in the formation of mutagens in the reaction of glucose/glycine/creatinine.

Two classes of the free radical Maillard intermediates, the pyrazine cation radical and the carbon-centered radicals, are detected in the reaction of Glc (glucose)/Gly (glycine) by electron spin resonance and spin trapping technique. Profile of the generation of the pyrazine cation radical in the reaction with different ratios of the reactants was found to be similar to that of the formation of mutagens in the subsequent reaction with creatinine. By contrast, profile of the generation of the carbon-centered radicals was not consistent with that of the mutagen formation. Thiol antioxidants and unsaturated fatty acids (or their esters) effectively scavenged the pyrazine cation radical generated in the reaction of Glc/Gly, and inhibited the formation of the mutagens in the reaction of Glc/Gly and creatinine. Ethanol, a sulfide and a saturated fatty acid were not effective to scavenge the pyrazine cation radical and did not inhibit the mutagen formation. The pyrazine cation radical rather than the carbon-centered radicals may play an important role in the mutagen formation. Thiol antioxidants and unsaturated fatty acids can be evaluated as inhibitors of the pyrazine cation radical-derived formation of the mutagens.

Effective inhibition by beta-carotene of cellular DNA breaking induced by peroxynitrous acid.

Peroxynitrous acid synthesized by reaction of hydrogen peroxide and nitrite and generated from 3-morpholinosydononimine (SIN-1) induced cellular DNA breaking of human promyelocytic leukemia HL-60 cells in phosphate buffer (pH 7.5) as assessed by alkaline single cell gel electrophoresis (comet) assay and quantification of comet types. Ascorbate and Trolox inhibited cellular DNA breaking induced by peroxynitrous acid, but the concentrations of these antioxidants required for effective inhibition was about 50-fold higher than that of peroxynitrous acid. beta-Carotene protected DNA breaking by peroxynitrous acid in 20% tetrahydrofuran-phosphate buffer (pH 7.5) much more effectively than ascorbate and Trolox. The concentrations of beta-carotene required for effective inhibition was lower than the concentration of peroxynitrous acid.