Reduced expression of the CDK inhibitor p27(KIP1) in rat two-stage bladder carcinogenesis and its association with expression profiles of p21(WAF1/Cip1) and p53.
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The cyclin-dependent kinase (CDK) inhibitor p27(KIP1) exerts its growth suppressive effects by targeting the cyclin-CDK complexes. Reduced protein levels of p27(KIP1) have been reported in numerous human cancers and this has been attributed to increased degradation. However, few reports have addressed the significance of p27(KIP1) expression in chemical carcinogenesis of rodents. In a rat two-stage urinary bladder carcinogenesis model, with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) initiation followed by promotion with sodium L-ascorbate (Na-AsA), we evaluated the expression of p27(KIP1) protein using immunohistochemistry during various stages of urinary bladder carcinogenesis. In addition, we evaluated the mRNA expression profiles for p27(KIP1), p21(WAF1/Cip1) and p53 in tumors. Fisher 344 rats were initiated with 0.05% BBN in the drinking water for 4 weeks and then administered 5% Na-AsA in the diet. Immunohistochemical examination revealed p27(KIP1) protein to be constitutively expressed in normal urothelium, simple hyperplasia and in most papillary and nodular (PN) hyperplasias and small papillomas, but diminished or absent in large papillomas and in transitional cell carcinomas. An inverse correlation between expression of p27(KIP1) and cell proliferation was generally observed. Quantitation of mRNA by multiplex reverse transcription-PCR showed a significant downregulaton of p27(KIP1), p21(WAF1/Cip1) and p53 mRNA in tumors. More than 50% reduction in p27(KIP1) mRNA expression was observed in 42 and 47% of tumors at weeks 18 and 24, respectively; similar reduction in p21(WAF1/Cip1) mRNA expression was observed in 58 and 73% of tumors at weeks 18 and 24, and in p53 mRNA expression in 50 and 73% of tumors at weeks 18 and 24, respectively. None of the 25 tumors we examined by PCR-single-strand conformational polymorphism analysis had p53 mutations. These data imply that abnormal down-regulation of p27(KIP1), p21(WAF1/Cip1) and/or p53 in tumor cells may contribute to the malignant progression of tumors during rat two-stage bladder carcinogenesis. (+info)
Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of procyanidin B5-3'-gallate as the most effective antioxidant constituent.
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Procyanidins present in grape seeds are known to exert anti-inflammatory, anti-arthritic and anti-allergic activities, prevent skin aging, scavenge oxygen free radicals and inhibit UV radiation-induced peroxidation activity. Since most of these events are associated with the tumor promotion stage of carcinogenesis, these studies suggest that grape seed polyphenols and the procyanidins present therein could be anticarcinogenic and/or anti-tumor-promoting agents. Therefore, we assessed the anti-tumor-promoting effect of a polyphenolic fraction isolated from grape seeds (GSP) employing the 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol 13-acetate (TPA)-promoted SENCAR mouse skin two-stage carcinogenesis protocol as a model system. Following tumor initiation with DMBA, topical application of GSP at doses of 0.5 and 1.5 mg/mouse/application to the dorsal initiated mouse skin resulted in a highly significant inhibition of TPA tumor promotion. The observed anti-tumor-promoting effects of GSP were dose dependent and were evident in terms of a reduction in tumor incidence (35 and 60% inhibition), tumor multiplicity (61 and 83% inhibition) and tumor volume (67 and 87% inhibition) at both 0.5 and 1.5 mg GSP, respectively. Based on these results, we directed our efforts to separate and identify the individual polyphenols present in GSP and assess their antioxidant activity in terms of inhibition of epidermal lipid peroxidation. Employing HPLC followed by comparison with authentic standards for retention times in HPLC profiles, physiochemical properties and spectral analysis, nine individual polyphenols were identified as catechin, epicatechin, procyanidins B1-B5 and C1 and procyanidin B5-3'-gallate. Five of these individual polyphenols with evident structural differences, namely catechin, procyanidin B2, procyanidin B5, procyanidin C1 and procyanidin B5-3'-gallate, were assessed for antioxidant activity. All of them significantly inhibited epidermal lipid peroxidation, albeit to different levels. A structure-activity relationship study showed that with an increase in the degree of polymerization in polyphenol structure, the inhibitory potential towards lipid peroxidation increased. In addition, the position of linkage between inter-flavan units also influences lipid peroxidation activity; procyanidin isomers with a 4-6 linkage showed stronger inhibitory activity than isomers with a 4-8 linkage. A sharp increase in the inhibition of epidermal lipid peroxidation was also evident when a gallate group was linked at the 3'-hydroxy position of a procyanidin dimer. Procyanidin B5-3'-gallate showed the most potent antioxidant activity with an IC(50) of 20 microM in an epidermal lipid peroxidation assay. Taken together, for the first time these results show that grape seed polyphenols possess high anti-tumor-promoting activity due to the strong antioxidant effect of procyanidins present therein. In summary, grape seed polyphenols in general, and procyanidin B5-3'-gallate in particular, should be studied in more detail to be developed as cancer chemopreventive and/or anticarcinogenic agents. (+info)
Cigarette smoke induces direct DNA damage in the human B-lymphoid cell line Raji.
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Human lymphoid cells (Raji) were exposed to water-soluble compounds from cigarette smoke (CS) generated in a smoking machine. DNA damage, as detected by alkaline single-cell microelectrophoresis (COMET assay), was induced in a time- and concentration-dependent manner in the cells. Most of the rapidly induced DNA damage was attributable to direct-acting compounds since cytochrome P450-related metabolic activities (ethoxy- and pentoxyresorufin-O-deethylases and coumarin-7-hydroxylase) were absent or very low. In addition, induction of DNA damage could be inhibited only slightly by beta-naphthoflavone and coumarin. Vitamin C enhanced DNA damage in Raji cells probably by redox cycling of catechol and hydroquinone present in CS implicating reactive oxygen intermediates as another source of DNA damage. N-acetylcysteine, a radical scavenger and glutathione precursor, reduced DNA damage in Raji cells when exposure to CS was followed by 2 h post-incubation in culture medium. Unrepaired DNA damage caused by CS persisted longer than gamma-irradiation-induced DNA damage. Among the CS constituents, acrolein, but not formaldehyde and acetaldehyde, induced DNA damage although less intensely than CS itself. At 50 and 100 microM concentrations, acrolein also inhibited repair of gamma- irradiation-induced DNA damage in the COMET assay. Inhibition of DNA synthesis by acrolein at 50 microM was demonstrated by an immunochemical assay for bromo-deoxyuridine incorporation; however, inhibition of a representative repair enzyme, 8-oxoguanosine hydrolase, by either CS or acrolein was not observed. The present results further confirm the presence of direct-acting genotoxic components and inhibitors of DNA repair in the gas phase of tobacco smoke, that may contribute to DNA damage and smoking-associated cancers of the upper aerodigestive tract. (+info)
The role of membrane fluidity changes and thiobarbituric acid-reactive substances production in the inhibition of cerebral cortex Na+/K+-ATPase activity.
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Lipid peroxidation of rat cerebral cortex membranes was induced by Fe2+/ADP and ascorbate. The rate of Na+/K(+)-ATPase inhibition was correlated with the increase of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD) and with membrane fluidity changes. Our data showed that membrane fluidity changes (evaluated by fluorescence steady-state anisotropy measurements) can participate in Na+/K(+)-ATPase inhibition during the initial period of lipid peroxidation process, whereas during the following period the enzyme inhibition correlates only with TBARS and CD production. (+info)
Compromised concentrations of ascorbate in fluid lining the respiratory tract in human subjects after exposure to ozone.
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OBJECTIVES: Ozone (O3) imposes an oxidative burden on the lung in two ways. Firstly, directly as a consequence of its oxidising character during exposure, and secondly, indirectly by engendering inflammation. In this study the second pathway was considered by ascertaining the impact of O3 on the redox state of the fluid lining the respiratory tract 6 hours after challenge. METHODS: Nine subjects were exposed in a double blind crossover control trial to air and 200 ppb O3 for 2 hours with an intermittent exercise and rest protocol. Blood samples were obtained and lung function (forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1)) assessed before, immediately after, and 6 hours after exposure. Bronchoalveolar lavage (BAL) was performed 6 hours after challenge. Inflammation was assessed in BAL fluid (total and differential cell counts, plus myeloperoxidase concentrations), and plasma and BAL fluid redox state were determined by measuring concentrations of antioxidants and markers of oxidative damage. RESULTS: Neutrophil numbers in BAL fluid increased 2.2-fold (p = 0.07) 6 hours after exposure and this was accompanied by increased myeloperoxidase concentrations in BAL fluid (p = 0.08). On the other hand, BAL fluid macrophage and lymphocyte numbers decreased 2.5-fold (p = 0.08) and 3.1-fold (p = 0.08), respectively at this time. Of the antioxidants examined, only ascorbate in BAL fluid was affected by O3, falling in all subjects relative to air values (0.1 (0.0-0.3) v 0.3 (0.2-1.2) mumol/l (p = 0.008)). A marginal decrease in plasma ascorbate was also detected at this time (p < 0.05). Although the decrease in macrophage numbers seemed to be causally related to the increase in neutrophils (R = -0.79), myeloperoxidase concentrations (R = -0.93) and ascorbate concentrations (R = 0.6), no clear associations were apparent between ascorbate changes and neutrophils or myeloperoxidase concentration after O3. CONCLUSIONS: Ascorbate in the fluid lining the respiratory tract is depleted as a consequence of O3 exposure at 6 hours after exposure. This was contemporaneous with, although not quantitatively related to the increase in neutrophil numbers and myeloperoxidase concentrations. Decreased macrophage numbers 6 hours after O3 related to the degree of neutrophilic inflammation with populations conserved where ascorbate concentration in the fluid lining the respiratory tract were high after exposure. These results imply that ascorbate has a critical protective role against inflammatory oxidative stress induced by O3. (+info)
The effect of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa.
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The aim of this study was to determine the effects of supplementation with ascorbate and alpha-tocopherol, both singly and in combination, during sperm preparation on subsequent sperm DNA integrity, induced DNA damage and reactive oxygen species (ROS) generation. Semen samples with normozoospermic and asthenozoospermic profiles (n = 15 for each control and antioxidant group) were prepared by Percoll density centrifugation (95.0-47.5%) where the medium had been supplemented with these antioxidants to a number of different concentrations, all within physiological levels. Controls were included which had no ascorbate or alpha-tocopherol added. DNA damage was induced using hydrogen peroxide (H(2)O(2)) and DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay, while ROS generation was measured using chemiluminescence. Addition of ascorbate to sperm preparation medium did not affect baseline DNA integrity but did provide sperm with complete protection against H(2)O(2)-induced DNA damage. Generation of H(2)O(2)-induced ROS was also significantly reduced after treatment with ascorbate, although baseline levels were unaffected by this antioxidant. Supplementation of sperm preparation medium with alpha-tocopherol did not influence baseline DNA integrity but provided sperm with dose-dependent protection against H(2)O(2)-induced DNA damage. Generation of H(2)O(2)-induced ROS was significantly reduced after treatment with alpha-tocopherol, although baseline ROS levels were unaffected by this antioxidant. Addition of both ascorbate and alpha-tocopherol in combination to sperm preparation medium actually induced DNA damage and intensified the damage induced by H(2)O(2), however, H(2)O(2)-induced ROS production was significantly reduced in a dose-dependent manner by supplementation with both vitamins. (+info)
Human sperm DNA integrity assessed by the Comet and ELISA assays.
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DNA integrity in sperm is essential for the accurate transmission of genetic information and therefore the maintenance of good health in future generations. The ELISA and Comet assays, two techniques that detect DNA damage in cells, are compared in this study of DNA integrity in human sperm. Both techniques rely on alkaline unwinding for the release of single strands of DNA from the nucleus. The ELISA detects single strands immunochemically whereas the Comet assay measures single strands drawn out by electrophoresis, stained with ethidium bromide and quantified by image analysis. The two techniques, both modified for use with sperm, detect similar levels of baseline DNA damage along with similar dose-dependent patterns of induced damage by X-ray irradiation at 10 and 30 Gy (P < 0.05). The assays are also comparable in the detection of a significant protective effect by ascorbic acid (300 and 600 microM) and alpha-tocopherol (30 and 60 microM) on DNA integrity, both at baseline levels and following X-ray irradiation (p < 0.01). The advantages and disadvantages of each technique are discussed. (+info)
Effects of Maillard reaction products on the oxidative cleavage and polymerization of protein under ascorbic acid-transition metal system.
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This study investigated the effects of Maillard reaction products (MRPs) on the oxidative cleavage and polymerization of BSA (bovine serum albumin) in an aqueous system. In L-ascorbic acid (AsA) and Cu(II) or Fe(III) reaction system, 50-60% of BSA was cleaved under physiological conditions (37 degrees C, pH 7.2). The oxidative cleavage induced by AsA-Cu(II) system was suppressed to the extent of 32-86% by model melanoidins or brown pigments from amino acids and foodstuffs. In the AsA-Fe(III) system, the oxidative cleavage was inhibited to the extent of 45-93% by melanoidins and brown pigments. However, this cleavage was promoted by amino acid Amadori rearrangement products and brown pigment from soy paste. Therefore, MRPs show both suppression and promotion activity on oxidative cleavage of BSA in the system of AsA and a transition metal. The quantity of Amadori rearrangement moiety (ARM) in melanoidins from Lysine and brown pigments molecules from foods was also measured. From these data, it was estimated that the suppression and/or promotion of oxidative cleavage of BSA did not only depend on the quantity of ARM, but also depended on the chemical structure of ARM in melanoidins or brown pigments. (+info)