Helen R. Griffiths

Vitamin C exhibits pro-oxidant properties

Vitamin C is marketed as a dietary supplement, partly because of its ‘antioxidant’ properties. However, we report here that vitamin C administered as a dietary supplement to healthy humans exhibits a pro-oxidant, as well as an antioxidant, effect in vivo.

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function.

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

Monocytes in coronary artery disease and atherosclerosis: where are we now?

Despite improvements in interventional and pharmacological therapy of atherosclerotic disease, it is still the leading cause of death in the developed world. Hence, there is a need for further development of effective therapeutic approaches. This requires better understanding of the molecular mechanisms and pathophysiology of the disease. Atherosclerosis has long been identified as having an inflammatory component contributing to its pathogenesis, whereas the available therapy primarily targets hyperlipidemia and prevention of thrombosis. Notwithstanding a pleotropic anti-inflammatory effect to some therapies, such as acetyl salicylic acid and the statins, none of the currently approved medicines for management of either stable or complicated atherosclerosis has inflammation as a primary target. Monocytes, as representatives of the innate immune system, play a major role in the initiation, propagation, and progression of atherosclerosis from a stable to an unstable state. Experimental data support a role of monocytes in acute coronary syndromes and in outcome post-infarction; however, limited research has been done in humans. Analysis of expression of various cell surface receptors allows characterization of the different monocyte subsets phenotypically, whereas downstream assessment of inflammatory pathways provides an insight into their activity. In this review we discuss the functional role of monocytes and their different subpopulations in atherosclerosis, acute coronary syndromes, cardiac healing, and recovery with an aim of critical evaluation of potential future therapeutic targets in atherosclerosis and its complications. We will also discuss technical difficulties of delineating different monocyte subpopulations, understanding their differentiation potential and function.

Molecular and Cellular Effects of Ultraviolet Light-Induced Genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earths stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

Novel repair action of vitamin C upon in vivo oxidative DNA damage

There appears to be a paucity of data examining the effect of dietary antioxidants on levels of oxidative DNA damage in vivo, limiting evidence‐based assessment of antioxidant efficacy, mechanisms and recommendation for optimal intake. We have examined levels of 8‐oxo‐2′‐deoxyguanosine (8‐oxodG) in mononuclear cell DNA, serum and urine from subjects undergoing supplementation with 500 mg/day vitamin C. Significant decreases in DNA levels of 8‐oxodG were seen, correlating strongly with increases in plasma vitamin C concentration. Furthermore we established a timecourse for sequential, significant increases in serum and urinary 8‐oxodG levels. These results illustrate, for the first time in humans, the kinetics of 8‐oxodG removal and processing in vivo, suggesting a role for vitamin C in the regulation of DNA repair enzymes and thereby demonstrating a non‐scavenging antioxidant effect.

8-Hydroxydeoxyguanosine. A marker of oxidative DNA damage in systemic lupus erythematosus

8‐Hydroxydeoxyguanosine (80HDG) is a specific marker of oxidative damage to DNA. We have observed that patients with SLE (systemic lupus erythematosus), have undetectable levels of urinary 8OHDG by HPLC. Further analysis by GC‐MS confirmed that levels of 8OHDG in SLE urine were 103‐fold lower than in an age‐ and sex‐matched control group. Experiments utilising cultures of SLE and normal lymphocytes exposed to H2O2 confirmed the impaired ability of SLE lymphocytes to repair 8OHDG. We subsequently observed in SLE patients that 8OHDG had accumulated in low molecular weight DNA associated with circulating immune complexes. We suggest that oxygen radicals may induce pathology in SLE by maintaining the presence of an antigenic form of DNA in the circulation.

Statin use in rheumatoid arthritis in relation to actual cardiovascular risk: evidence for substantial undertreatment of lipid-associated cardiovascular risk?

Background Cardiovascular disease (CVD) is partially attributed to traditional cardiovascular risk factors, which can be identified and managed based on risk stratification algorithms (Framingham Risk Score, National Cholesterol Education Program, Systematic Cardiovascular Risk Evaluation and Reynolds Risk Score). We aimed to (a) identify the proportion of at risk patients with rheumatoid arthritis (RA) requiring statin therapy identified by conventional risk calculators, and (b) assess whether patients at risk were receiving statins. Methods Patients at high CVD risk (excluding patients with established CVD or diabetes) were identified from a cohort of 400 well characterised patients with RA, by applying risk calculators with or without a ×1.5 multiplier in specific patient subgroups. Actual statin use versus numbers eligible for statins was also calculated. Results The percentage of patients identified as being at risk ranged significantly depending on the method, from 1.6% (for 20% threshold global CVD risk) to 15.5% (for CVD and cerebrovascular morbidity and mortality) to 21.8% (for 10% global CVD risk) and 25.9% (for 5% CVD mortality), with the majority of them (58.1% to 94.8%) not receiving statins. The application of a 1.5 multiplier identified 17% to 78% more at risk patients. Conclusions Depending on the risk stratification method, 2% to 26% of patients with RA without CVD have sufficiently high risk to require statin therapy, yet most of them remain untreated. To address this issue, we would recommend annual systematic screening using the nationally applicable risk calculator, combined with regular audit of whether treatment targets have been achieved.

The anti-inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species.

The mechanism of action by which methotrexate (MTX) exerts its anti‐inflammatory and immunosuppressive effects remains unclear. The aim of this study is to investigate the hypothesis that MTX exerts these effects via the production of reactive oxygen species (ROS). Addition of MTX (100 nM–10 μM) to U937 monocytes induced a time and dose dependent increase in cytosolic peroxide [peroxide]cyt from 6–16 h. MTX also caused corresponding monocyte growth arrest, which was inhibited (P<0.05) by pre‐treatment with N‐acetylcysteine (NAC; 10 mM) or glutathione (GSH; 10 mM). In contrast, MTX induction of [peroxide]cyt in Jurkat T cells was more rapid (4 h; P<0.05), but was associated with significant apoptosis at 16 h at all doses tested (P<0.05) and was significantly inhibited by NAC or GSH (P<0.05). MTX treatment of monocytes (10 nM–10 μM) for 16 h significantly reduced total GSH levels (P<0.05) independently of dose (P>0.05). However, in T‐cells, GSH levels were significantly elevated following 30 nM MTX treatment (P<0.05) but reduced by doses exceeding 1 μM compared to controls (P<0.05). MTX treatment significantly reduced monocyte adhesion to 5 h and 24 h LPS (1 μg ml−1) activated human umbilical vein endothelial cells (HUVEC; P<0.05) but not to resting HUVEC. Pre‐treatment with GSH prevented MTX‐induced reduction in adhesion. In conclusion, ROS generation by MTX is important for cytostasis in monocytes and cytotoxicity T‐cells. Furthermore, MTX caused a reduction in monocyte adhesion to endothelial cells, where the mechanism of MTX action requires the production of ROS. Therefore its clinical efficacy can be attributed to multiple targets.

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C‐reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non‐smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 µg/ml for 0–60 min at 37°C and analysed for (a) CD11b, PECAM‐1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 µg/ml; 0–24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS‐activated HUVEC (0–5 h). However, the adhesion of CRP‐treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C‐reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.