Ascorbate and α-tocopherol differentially modulate reactive oxygen species generation by neutrophils in response to FcγR and TLR agonists

Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 μM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects in vivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function. © The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and d-α-tocopherol (10 μM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-κB (NF-κB), through inhibition of phosphorylation of the NF-κB inhibitor protein (I-κB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 μM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 μM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 μM) and D609 (200 μM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 μM) and NSC23766 (10 μM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 μM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 μM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with d-α-tocopherol (1 mg kg- 1) attenuated protein degradation and increased protein synthesis in skeletal muscle. © 2007 Elsevier Inc. All rights reserved.

The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1α(βS*, δS*), 2α, 6α, 8β-(R*), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1∝(ßS*,dS*),2∝,6a∝,8ß-(R*),8a∝]]-1,2,6,7,8,8a-hexahydro-ß,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 ± 1.9% versus 16.6 ± 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 ± 2.9% versus 23.4 ± 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC50, 6.37 ± 0.37) compared with both vehicle-treated (-logEC50, 5.81 ± 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 ± 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 ± 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.

Chemical specificity in REDOX-responsive materials:the diverse effects of different Reactive Oxygen Species (ROS) on polysulfide nanoparticles

REDOX responsive (nano)materials typically exhibit chemical changes in response to the presence and concentration of oxidants/reductants. Due to the complexity of biological environments, it is critical to ascertain whether the chemical response may depend on the chemical details of the stimulus, in addition to its REDOX potential, and whether chemically different responses can determine a different overall performance of the material. Here, we have used oxidation-sensitive materials, although these considerations can be extended also to reducible ones. In particular, we have used poly(propylene sulfide) (PPS) nanoparticles coated with a PEGylated emulsifier (Pluronic F127); inter alia, we here present also an improved preparative method. The nanoparticles were exposed to two Reactive Oxygen Species (ROS) typically encountered in inflammatory reactions, hydrogen peroxide (H2O2) and hypochlorite (ClO−); their response was evaluated with a variety of techniques, including diffusion NMR spectroscopy that allowed to separately characterize the chemically different colloidal species produced. The two oxidants triggered a different chemical response: H2O2 converted sulfides to sulfoxides, while ClO− partially oxidized them further to sulfones. The different chemistry correlated to a different material response: H2O2 increased the polarity of the nanoparticles, causing them to swell in water and to release the surface PEGylated emulsifier; the uncoated oxidized particles still exhibited very low toxicity. On the contrary, ClO− rapidly converted the nanoparticles into water-soluble, depolymerized fragments with a significantly higher toxicity. The take-home message is that it is more correct to discuss ‘smart’ materials in terms of an environmentally specific response to (REDOX) stimuli. Far from being a problem, this could open the way to more sophisticated and precisely targeted applications.

Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.

New insights into plant salt acclimation: the roles of vesicle trafficking and reactive oxygen species signalling in mitochondria and the endomembrane system

In this study, we investigated the cellular and molecular mechanisms that regulate salt acclimation. The main objective was to obtain new insights into the molecular mechanisms that control salt acclimation. Therefore, we carried out a multidisciplinary study using proteomic, transcriptomic, subcellular and physiological techniques. We obtained a Nicotiana tabacum BY-2 cell line acclimated to be grown at 258 mM NaCl as a model for this study. The proteomic and transcriptomic data indicate that the molecular response to stress (chaperones, defence proteins, etc.) is highly induced in these salt-acclimated cells. The subcellular results show that salt induces sodium compartmentalization in the cell vacuoles and seems to be mediated by vesicle trafficking in tobacco salt-acclimated cells. Our results demonstrate that abscisic acid (ABA) and proline metabolism are crucial in the cellular signalling of salt acclimation, probably regulating reactive oxygen species (ROS) production in the mitochondria. ROS may act as a retrograde signal, regulating the cell response. The network of endoplasmic reticulum and Golgi apparatus is highly altered in salt-acclimated cells. The molecular and subcellular analysis suggests that the unfolded protein response is induced in salt-acclimated cells. Finally, we propose that this mechanism may mediate cell death in salt-acclimated cells.

The magnitude of the snow-sourced reactive nitrogen flux to the boundary layer in the Uintah Basin, Utah, USA

Reactive nitrogen (Nr=NO, NO2, HONO) and volatile organic carbon emissions from oil and gas extraction activities play a major role in wintertime ground-level ozone exceedance events of up to 140 ppb in the Uintah Basin in eastern Utah. Such events occur only when the ground is snow covered, due to the impacts of snow on the stability and depth of the boundary layer and ultraviolet actinic flux at the surface. Recycling of reactive nitrogen from the photolysis of snow nitrate has been observed in polar and mid-latitude snow, but snow-sourced reactive nitrogen fluxes in mid-latitude regions have not yet been quantified in the field. Here we present vertical profiles of snow nitrate concentration and nitrogen isotopes (δ15N) collected during the Uintah Basin Winter Ozone Study 2014 (UBWOS 2014), along with observations of insoluble light-absorbing impurities, radiation equivalent mean ice grain radii, and snow density that determine snow optical properties. We use the snow optical properties and nitrate concentrations to calculate ultraviolet actinic flux in snow and the production of Nr from the photolysis of snow nitrate. The observed δ15N(NO3-) is used to constrain modeled fractional loss of snow nitrate in a snow chemistry column model, and thus the source of Nr to the overlying boundary layer. Snow-surface δ15N(NO3-) measurements range from -5‰ to 10‰ and suggest that the local nitrate burden in the Uintah Basin is dominated by primary emissions from anthropogenic sources, except during fresh snowfall events, where remote NOx sources from beyond the basin are dominant. Modeled daily-averaged snow-sourced Nr fluxes range from 5.6-71x107 molec cm-2 s-1 over the course of the field campaign, with a maximum noon-time value of 3.1x109 molec cm-2 s-1. The top-down emission estimate of primary, anthropogenic NOx in the Uintah and Duchesne counties is at least 300 times higher than the estimated snow NOx emissions presented in this study. Our results suggest that snow-sourced reactive nitrogen fluxes are minor contributors to the Nr boundary layer budget in the highly-polluted Uintah Basin boundary layer during winter 2014.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX.

Cytoplasmic Irradiation Induces Mitochondrial-Dependent 53BP1 Protein Relocalization in Irradiated and Bystander Cells

The accepted paradigm for radiation effects is that direct DNA damage via energy deposition is required to trigger the downstream biological consequences. The radiation-induced bystander effect is the ability of directly irradiated cells to interact with their nonirradiated neighbors, which can then show responses similar to those of the targeted cells. p53 binding protein 1 (53BP1) forms foci at DNA double-strand break sites and is an important sensor of DNA damage. This study used an ionizing radiation microbeam approach that allowed us to irradiate specifically the nucleus or cytoplasm of a cell and quantify response in irradiated and bystander cells by studying ionizing radiation-induced foci (IRIF) formation of 53BP1 protein. Our results show that targeting only the cytoplasm of a cell is capable of eliciting 53BP1 foci in both hit and bystander cells, independently of the dose or the number of cells targeted. Therefore, direct DNA damage is not required to trigger 53BP1 IRIF. The use of common reactive oxygen species and reactive nitrogen species (RNS) inhibitors prevent the formation of 53BP1 foci in hit and bystander cells. Treatment with filipin to disrupt membrane-dependent signaling does not prevent the cytoplasmic irradiation-induced 53BP1 foci in the irradiated cells, but it does prevent signaling to bystander cells. Active mitochondrial function is required for these responses because pseudo-rho(0) cells, which lack mitochondrial DNA, could not produce a bystander signal, although they could respond to a signal from normal rho(+) cells.

The contribution of N₂O₃ to the cytotoxicity of the nitric oxide donor DETA/NO: an emerging role for S-nitrosylation

The relationship between the biological activity of NO and its chemistry is complex. The objectives of this study were to investigate the influence of oxygen tension on the cytotoxicity of the NO• donor DETA/NO and to determine the effects of oxygen tension on the key RNS (reactive nitrogen species) responsible for any subsequent toxicity. The findings presented in this study indicate that the DETA/NO-mediated cytotoxic effects were enhanced under hypoxic conditions. Further investigations revealed that neither ONOO⁻ (peroxynitrite) nor nitroxyl was generated. Fluorimetric analysis in the presence of scavengers suggest for the first time that another RNS, dinitrogen trioxide may be responsible for the cytotoxicity with DETA/NO. Results showed destabilization of HIF (hypoxia inducible factor)-1α and depletion of GSH levels following the treatment with DETA/NO under hypoxia, which renders cells more susceptible to DETA/NO cytotoxicity, and could account for another mechanism of DETA/NO cytotoxicity under hypoxia. In addition, there was significant accumulation of nuclear p53, which showed that p53 itself might be a target for S-nitrosylation following the treatment with DETA/NO. Both the intrinsic apoptotic pathway and the Fas extrinsic apoptotic pathway were also activated. Finally, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is another important S-nitrosylated protein that may possibly play a key role in DETA/NO-mediated apoptosis and cytotoxicity. Therefore this study elucidates further mechanisms of DETA/NO mediated cytotoxicity with respect to S-nitrosylation that is emerging as a key player in the signalling and detection of DETA/NO-modified proteins in the tumour microenvironment.

Interactions of a non-thermal atmospheric pressure plasma effluent with PC-3 prostate cancer cells

The effect of a radio-frequency driven, microscale non thermal atmospheric pressure plasma jet operated in helium with vol. 0.3% molecular oxygen gas admixture, on PC-3 prostate cancer cells has been investigated. The viability of cells exposed to the plasma was found to decrease with increasing plasma exposure time, with apoptosis through caspase and PARP cleavage being observed. High concentrations of nitrite and nitrate were detected in growth media exposed to the plasma and were found to increase in a time dependent manner post exposure. This indicates a slow release of reactive nitrogen species into the growth media, which is likely to influence cellular response to plasma exposure.

Free radicals, metals and antioxidants in oxidative stress-induced cancer

Oxygen-free radicals, more generally known as reactive oxygen species (ROS) along with reactive nitrogen species (RNS) are well recognised for playing a dual role as both deleterious and beneficial species. The "two-faced" character of ROS is substantiated by growing body of evidence that ROS within cells act as secondary messengers in intracellular signalling cascades, which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. The cumulative production of ROS/RNS through either endogenous or exogenous insults is termed oxidative stress and is common for many types of cancer cell that are linked with altered redox regulation of cellular signalling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (8-OH-G) have been noted in various tumours, strongly implicating such damage in the etiology of cancer. It appears that the DNA damage is predominantly linked with the initiation process. This review examines the evidence for involvement of the oxidative stress in the carcinogenesis process. Attention is focused on structural, chemical and biochemical aspects of free radicals, the endogenous and exogenous sources of their generation, the metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel)-mediated formation of free radicals (e.g. Fenton chemistry), the DNA damage (both mitochondrial and nuclear), the damage to lipids and proteins by free radicals, the phenomenon of oxidative stress, cancer and the redox environment of a cell, the mechanisms of carcinogenesis and the role of signalling cascades by ROS; in particular. ROS activation of AP-1 (activator protein) and NF-kappa B (nuclear factor kappa B) signal transduction pathways, which, in turn lead to the transcription of genes involved in cell growth regulatory pathways. The role of enzymatic (superoxide dismutase (Cu. Zn-SOD. Mn-SOD), catalase, glutathione peroxidase) and non-enzymatic antioxidants (Vitamin C, Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic acid), flavonoids, selenium and others) in the process of careinogenesis as well as the antioxidant interactions with various regulatory factors, including Ref-1, NF-kappa B, AP-1 are also reviewed. 2006 Elsevier Ireland Ltd. All rights reserved.

The reaction of flavanols with nitrous acid protects against N-nitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth

Studies have suggested that diets rich in polyphenols Such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D I levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract. (c) 2005 Elsevier Inc. All rights reserved.