Season, storage and extraction method impact on the phytochemical profile of Terminalia ivorensis.

BACKGROUND: Terminalia ivorensis (TI) is used in West African ethnomedicine for the treatment of conditions including ulcers, malaria and wounds. Despite its widespread use, the phytochemical profile of TI remains largely undetermined. This research investigated the effects of extraction method, season, and storage conditions on the phytochemical composition of TI to contribute towards understanding the potential benefits. METHODS: TI bark was collected in September 2014, September 2018 and February 2018 during the rainy or dry seasons in Eastern Region, Ghana. Samples were extracted sequentially with organic solvents (petroleum ether, chloroform, ethyl acetate and ethanol) or using water (traditional). Metabolites were identified by liquid chromatography-mass spectrometry/mass spectrometry and compared statistically by ANOVA. RESULTS: A total of 82 different phytochemicals were identified across all samples. A greater yield of the major phytochemicals (44%, p < 0.05) was obtained by water as compared with organic extraction. There was also a higher concentration of metabolites present in cold (63%, p < 0.05) compared with hot water extraction. A significantly (p < 0.05) higher number of phytochemicals were identified from TI collected in the dry (85%) compared to the rainy season (69%). TI bark stored for four years retained 84% of the major phytochemicals. CONCLUSION: This work provides important information on composition and how this is modified by growing conditions, storage and method of extraction informing progress on the development of TI as a prophylactic formulation or medicine.

The nutritional and cardiovascular health benefits of rapeseed oil-fed farmed salmon in humans are not decreased compared with those of traditionally farmed salmon: a randomized controlled trial.

PURPOSE: Farmed fish are increasingly raised on feeds containing vegetable oils, which affects their composition and possibly health properties. We investigated the effects of consuming farmed salmon, raised on different feeding regimes, on nutrient status and health outcomes in healthy subjects. METHODS: Salmon were grown on feeds containing mainly fish oil (FO) or rapeseed oil (RO), resulting in an eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) content of fillets of 2.1 or 0.9 g/100 g, respectively. In a randomized parallel controlled trial, 51 healthy subjects were allocated to consume 2 portions/week of FO salmon (n = 17), RO salmon (n = 17) or no additional salmon (Control, n = 17) as part of their habitual diet, for 18 weeks. We collected blood at 0, 9 and 18 weeks to measure omega-3 index (O3I) in red blood cells, plasma markers of cardiovascular risk, serum 25(OH)-vitamin D3 (25(OH)D3) and plasma trace elements. RESULTS: After 18 weeks, O3I was similarly increased in subjects consuming 2 portions/week of FO or RO salmon compared to control (both p < 0.05). Serum 25(OH)D3 was significantly higher, whereas plasma triacylglycerols were significantly lower in subjects consuming RO salmon compared to control (both p < 0.05). Heart rate was significantly lower in subjects consuming FO salmon after 9 weeks, compared to control (p < 0.01). Salmon consumption did not affect other markers. CONCLUSION: Consuming two portions/week of salmon raised on rapeseed oil rather than fish oil increased the O3I and vitamin D status, and decreased plasma triacylglycerols. These outcomes endorse opportunities for developing more sustainable feeds within aquaculture food systems. CLINICAL TRIAL REGISTRY: This trial was registered at clinicaltrials.gov as NCT01916434.

Folate deficiency promotes differentiation of vascular smooth muscle cells without affecting the methylation status of regulated genes.

Elevated serum homocysteine, an intermediate of cellular one-carbon metabolism, is an independent risk factor for cardiovascular disease (CVD). Folate deficiency increases serum homocysteine and may contribute to CVD progression. Vascular smooth muscle cells (VSMCs) regulate vascular contractility, but also contribute to repair processes in response to vascular injury. Nutritional deficiencies, like folate deficiency, are thought to impact on this phenotypic plasticity, possibly by epigenetic mechanisms. We have investigated the effect of folate deficiency on VSMCs in two cell culture systems representing early and late stages of smooth muscle cells differentiation. We find that folate deficiency promotes differentiation towards a more contractile phenotype as indicated by increased expression of respective marker genes. However, microarray analysis identified markers of striated muscle as the predominant gene expression change elicited by folate deficiency. These changes are not merely a reflection of cell cycle arrest, as foetal calf serum restriction or iron deficiency do not replicate the gene expression changes observed in response to folate deficiency. Folate deficiency only has a marginal effect on global DNA methylation. DNA methylation of CpG islands associated with genes regulated by folate deficiency remains unaffected. This supports our earlier findings in a mouse model system which also did not show any changes in global DNA methylation in response to folate and vitamin B6/B12 deficiency. These data suggest that folate deficiency enhances the expression of smooth muscle marker gene expression, promotes a shift towards a skeletal muscle phenotype, and does not regulate gene expression via DNA methylation.

Roadmap for investigating epigenome deregulation and environmental origins of cancer.

The interaction between the (epi)genetic makeup of an individual and his/her environmental exposure record (exposome) is accepted as a determinant factor for a significant proportion of human malignancies. Recent evidence has highlighted the key role of epigenetic mechanisms in mediating gene-environment interactions and translating exposures into tumorigenesis. There is also growing evidence that epigenetic changes may be risk factor-specific ("fingerprints") that should prove instrumental in the discovery of new biomarkers in cancer. Here, we review the state of the science of epigenetics associated with environmental stimuli and cancer risk, highlighting key developments in the field. Critical knowledge gaps and research needs are discussed and advances in epigenomics that may help in understanding the functional relevance of epigenetic alterations. Key elements required for causality inferences linking epigenetic changes to exposure and cancer are discussed and how these alterations can be incorporated in carcinogen evaluation and in understanding mechanisms underlying epigenome deregulation by the environment.

Effect of increasing fruit and vegetable intake by dietary intervention on nutritional biomarkers and attitudes to dietary change: a randomised trial.

PURPOSE: Low fruit and vegetable consumption is linked with an increased risk of death from vascular disease and cancer. The benefit of eating fruits and vegetables is attributed in part to antioxidants, vitamins and phytochemicals. Whether increasing intake impacts on markers of disease remains to be established. This study investigates whether increasing daily intake of fruits, vegetables and juices from low (approx. 3 portions), to high intakes (approx. 8 portions) impacts on nutritional and clinical biomarkers. Barriers to achieving the recommended fruit and vegetable intakes are also investigated. METHOD: In a randomised clinical trial, the participants [19 men and 26 women (39-58 years)] with low reported fruit, juice and vegetable intake (<3 portions/day) were randomised to consume either their usual diet or a diet supplemented with an additional 480 g of fruit and vegetables and fruit juice (300 ml) daily for 12 weeks. Nutritional biomarkers (vitamin C, carotenoids, B vitamins), antioxidant capacity and genomic stability were measured pre-intervention, at 4-, 8- and 12 weeks throughout the intervention. Samples were also taken post-intervention after a 6-week washout period. Glucose, homocysteine, lipids, blood pressure, weight and arterial stiffness were also measured. Intake of fruit, fruit juice and vegetables was reassessed 12 months after conducting the study and a questionnaire was developed to identify barriers to healthy eating. RESULTS: Intake increased significantly in the intervention group compared to controls, achieving 8.4 portions/day after 12 weeks. Plasma vitamin C (35%), folate (15%) and certain carotenoids [α-carotene (50%) and ß-carotene (70%) and lutein/zeaxanthin (70%)] were significantly increased (P < 0.05) in the intervention group. There were no significant changes in antioxidant capacity, DNA damage and markers of vascular health. Barriers to achieving recommended intakes of fruits and vegetables measured 12 months after the intervention period were amount, inconvenience and cost. CONCLUSION: While increasing fruit, juice and vegetable consumption increases circulating level of beneficial nutrients in healthy subjects, a 12-week intervention was not associated with effects on antioxidant status or lymphocyte DNA damage. TRIAL REGISTRATION: This trial was registered at Controlled-Trials.com; registration ISRCTN71368072.

Homocysteine, antioxidant micronutrients and late onset dementia.

PURPOSE: To distinguish between contributions to dementia made by homocysteine, folate, B12 and antioxidant micronutrients. METHODS: This is a follow-up study of a sample reported in 2002. Homocysteine was measured at baseline in 201 individuals born in 1921 and without dementia at age 77 years and followed up to age 88 years. Baseline macro- and micronutrient status was estimated from BMI, the MONICA food frequency questionnaire, plasma folate, B12 and, in a subgroup (N = 173), plasma antioxidant micronutrients. Time to dementia onset during follow-up was compared between participants grouped by homocysteine concentration using Cox regression. Model 1 adjusted for age, sex, childhood IQ, education, socioeconomic deprivation, presence of heart disease, hypertension, plasma folate and B12. In model 2 plasma, antioxidants were added to these covariables. RESULTS: During a mean follow-up of about 5 years, there were 39 incident dementia cases among 201 participants. In model 1, being in the highest homocysteine group (>14 µmol/L) was associated with a 234 % increased risk (HR 3.34, 95 % CI 1.16-9.57) of any dementia. After inclusion of plasma antioxidants in model 2, there were 32 incident dementia cases from a subsample (N = 173). Homocysteine >14 µmol was associated with a 272 % increased dementia risk (HR = 3.72, 95 % CI 1.06-13.08). CONCLUSIONS: High homocysteine increases the risk of dementia. The association between tHcy and dementia is independent of plasma folate, B12 and antioxidant micronutrient status.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Folate and cancer: how DNA damage, repair and methylation impact on colon carcinogenesis.

Inappropriate diet may contribute to one third of cancer deaths. Folates, a group of water-soluble B vitamins present in high concentrations in green, leafy vegetables, maintain DNA stability through their ability to donate one-carbon units for cellular metabolism. Folate deficiency has been implicated in the development of several cancers, including cancer of the colorectum, breast, ovary, pancreas, brain, lung and cervix. Generally, data from the majority of human studies suggest that people who habitually consume the highest level of folate, or with the highest blood folate concentrations, have a significantly reduced risk of developing colon polyps or cancer. However, an entirely protective role for folate against carcinogenesis has been questioned, and recent data indicate that an excessive intake of synthetic folic acid (from high-dose supplements or fortified foods) may increase human cancers by accelerating growth of precancerous lesions. Nonetheless, on balance, evidence from the majority of human studies indicates that dietary folate is genoprotective against colon cancer. Suboptimal folate status in humans is widespread. Folate maintains genomic stability by regulating DNA biosynthesis, repair and methylation. Folate deficiency induces and accelerates carcinogenesis by perturbing each of these processes. This review presents recent evidence describing how these mechanisms act, and interact, to modify colon cancer risk.

The influence of bisnaphthalimidopropyl polyamines on DNA instability and repair in Caco-2 colon epithelial cells.

Bisnaphthalimido compounds bis-intercalate to DNA via the major groove and are potentially potent cancer therapeutics. Previously, we incorporated natural polyamines as linkers connecting the two naphthalimido ring moieties to create a series of soluble bisnaphthalimidopropyl polyamines (BNIPPs). Here, extending earlier work on bisnaphthalimidopropylspermidine (BNIPSpd)-induced apoptosis in colon adenocarcinoma Caco-2 cells, we compare the cytotoxicity and genotoxicity of BNIPSpd relative to the spermine and oxaspermine derivatives, bisnaphthalimidopropylspermine (BNIPSpm) and bisnaphthalimidopropyloxaspermine (BNIPOSpm). The order of cytotoxicity after 24 h was BNIPSpd (IC(50) = 0.47 µM) > BNIPSpm (IC(50) = 10.04 µM) > BNIPOSpm (IC(50) >50 µM). After a 72-h BNIPOSpm exposure, an IC(50) = 10.25 µM was achieved. With 4-h exposure to BNIPSpd or BNIPSpm or 12-h exposure to BNIPOSpm, concentrations ≥1 µM induced a significant dose-dependent increase in DNA damage as measured by alkaline single-cell gel electrophoresis. The longer incubation times required for BNIPOSpm to induce DNA strand breaks reflect a slower rate of BNIPOSpm cellular distribution as monitored via BNIPP fluorescence within the cells. Moreover, exposure to a non-genotoxic concentration of BNIPSpd, BNIPSpm (0.1 µM for 4 h) or BNIPOSpm (0.1 µM for 12 h) induced a significant decrease in repair of oxidative DNA damage induced by hydrogen peroxide. In conclusion, BNIPP exposure in Caco-2 cells is associated with significant induction of DNA damage and inhibition of DNA repair at non-genotoxic concentrations. The latter is a novel consequence of BNIPP-cell interactions which adds to the spectrum of therapeutically relevant activities that may be exploited for the design and development of naphthalimide-based therapeutics.

Blood folate status and expression of proteins involved in immune function, inflammation, and coagulation: biochemical and proteomic changes in the plasma of humans in response to long-term synthetic folic acid supplementation.

We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

Rapid quantification of aortic lesions in apoE(-/-) mice.

The quantification of aortic lesions is an important end-point analysis for evaluating atherogenesis in mouse models of atherosclerosis. Morphometric methods involving the staining of aorta with a Sudan lysochrome followed by image analysis of the stained lesion area are commonly used. We have developed a more rapid method involving solubilisation of the stain retained by aortic lesions. In 2 separate studies, 5-week-old male apoE(-/-) and C57BL/6 wild-type (apoE(+/+)) mice were given a high fat (21%), Western-type diet for 13, 15 or 25 weeks. At study termination, the descending thoracic aorta (DA) and/or aortic arch (AA) were stained with Oil Red O (ORO). The incorporated stain was extracted using chloroform/methanol (2:1) solvent and quantified by spectrophotometry at 520 nm. In study 1 (13 weeks), ORO stain in the AA and DA of apoE(-/-) mice was 1.9 and 1.4 times higher than background staining of apoE(+/+) aorta tissue, respectively. At 15 and 25 weeks (study 2), ORO stain in the AA of apoE(-/-) mice was 1.9 and 2.5 times higher than the background, respectively. We conclude that the ORO solubilisation technique applied to AA samples is a very useful and rapid method for atherosclerotic lesion quantification.

Changes in vitamin biomarkers during a 2-year intervention trial involving increased fruit and vegetable consumption by free-living volunteers.

Trials in free-living populations involving increased consumption of fruit and vegetables are difficult to monitor. We evaluated biomarkers for assessing fruit and vegetable intake and compliance in a 2-year trial. Postmenopausal women were randomised to 300 g additional fruit and vegetables per d (n 66), placebo (n 70) or potassium citrate (n 140). They completed dietary checklists (3-monthly) and food diaries or FFQ (yearly). We measured whole-blood folate, plasma vitamin C and homocysteine (yearly), serum vitamin E and carotenoids (at 12 months) and urinary vitamin K metabolites (yearly). Plasma vitamin C was associated with fruit and vegetable intake at baseline (r +0.31; P < 0.01), remaining significant only for the non-fruit and vegetable group at 12 months (r +0.43; P < 0.01). For the fruit and vegetable group, vitamin C increased by 5.9 micromol/l (P = 0.07) but was not significantly associated with fruit and vegetable intake; vitamin E, beta-carotene and beta-cryptoxanthin were higher compared with the non-fruit and vegetable group (P < 0.05); and whole-blood folate and the urinary 5C-aglycone metabolite of vitamin K were associated with vegetable intake. For all participants plasma vitamin C increased with increasing fruit and vegetable intakes, reaching a plateau of 90-95 micromol/l at intakes>500 g/d, whereas whole-blood folate, beta-carotene and beta-cryptoxanthin continued to increase. Concentrations of vitamin C, folate and beta-cryptoxanthin were lower and the 7C-aglycone metabolite of vitamin K higher, in smokers compared with non-smokers. Suitable markers for monitoring fruit and vegetable compliance include beta-carotene and beta-cryptoxanthin. Plasma vitamin C and whole-blood folate may be suitable for monitoring intakes in populations but for monitoring compliance the former may be restricted to low intakes of fruit and vegetables and the latter to vegetable intake.

Folate, genomic stability and colon cancer: The use of single cell gel electrophoresis in assessing the impact of folate in vitro, in vivo and in human biomonitoring.

Intake of folate (vitamin B9) is strongly inversely linked with human cancer risk, particularly colon cancer. In general, people with the highest dietary intake of folate or with high blood folate levels are at a reduced risk (approx. 25%) of developing colon cancer. Folate acts in normal cellular metabolism to maintain genomic stability through the provision of nucleotides for DNA replication and DNA repair and by regulating DNA methylation and gene expression. Folate deficiency can accelerate carcinogenesis by inducing misincorporation of uracil into DNA, by increasing DNA strand breakage, by inhibiting DNA base excision repair capacity and by inducing DNA hypomethylation and consequently aberrant gene and protein expression. Conversely, increasing folate intake may improve genomic stability. This review describes key applications of single cell gel electrophoresis (the comet assay) in assessing genomic instability (misincorporated uracil, DNA single strand breakage and DNA repair capacity) in response to folate status (deficient or supplemented) in human cells in vitro, in rodent models and in human case-control and intervention studies. It highlights an adaptation of the SCGE comet assay for measuring genome-wide and gene-specific DNA methylation in human cells and colon tissue.

Caspase-independence and characterization of bisnaphthalimidopropyl spermidine induced cytotoxicity in HL60 cells.

Bisnaphthalimides are DNA intercalators of potential use as chemotherapeutics but for which the range of mechanism of action is only gradually being elucidated. Using human promyelocytic HL-60 cells, we extend characterization of the cytotoxicity of bisnaphthalimidopropylspermidine (BNIPSpd) and examine the relationship with caspase-activity. Within 4 h exposure, BNIPSpd (1-10 µM) induced significant DNA strand breakage. Evidence of apoptosis was progressive through the experimental period. Within 6 h, BNIPSpd increased the proportion of cells exhibiting plasma membrane phosphatidylserine exposure. Within 12 h, active caspase expression increased and was sustained with 5 and 10 µM BNIPSpd. Flow cytometric analysis revealed caspase activity in cells with and without damaged membranes. By 24 h, 5 and 10 µM BNIPSpd increased hypodiploid DNA content and internucleosomal DNA fragmentation (DNA ladders) typical of the later stages of apoptosis. 1 µM BNIPSpd exposure also increased hypodiploid DNA content by 48 h. Polyamine levels decreased by 24 h BNIPSpd exposure. The pan-caspase inhibitor, z-VAD-fmk, significantly decreased DNA degradation (hypodiploid DNA and DNA ladders) and cytotoxicity. Despite this, cell growth and viability remained significantly impaired. We propose that BNIPSpd cytotoxicity arises through DNA damage and not polyamine depletion and that cytotoxicity is dominated by but not dependent upon caspase driven apoptosis.

Berry phytochemicals, genomic stability and cancer: evidence for chemoprotection at several stages in the carcinogenic process.

Consumption of a diet high in plant-based foods is associated with a decreased risk of epithelial cell cancers at several sites. Cytoprotectants in fruits and vegetables include vitamins, minerals and numerous micronutrients. While there is little evidence uniquely linking berry consumption with lower cancer risk, berries contain high levels of compounds believed to reduce malignant transformation, including the polyphenol flavonoids and anthocyanins. There is strong and convincing evidence that berry extracts and berry phytochemicals modulate biomarkers of DNA damage and indicators of malignant transformation in vitro and in vivo. Data from numerous cell culture and animal models indicate that berry components such as the anthocyanins are potent anticarcinogenic agents and are protective against genomic instability at several sites in the carcinogenic pathway. Anticarcinogenic mechanisms include modulation of carcinogen activation and detoxification, decreased DNA binding of the carcinogen, inhibition of oxidative DNA damage, alteration in cell signalling and malignant transformation and inhibition of cell invasiveness and metastasis. Exactly which berry constituents are cytoprotective remains uncertain and in the majority of in vitro and in vivo studies the concentration of extract or phytochemical employed is non-nutritional. Evidence for an anticarcinogenic effect in human studies is weak.

Kaempferol induced inhibition of HL-60 cell growth results from a heterogeneous response, dominated by cell cycle alterations.

Dietary flavonoids may be exploitable as chemotherapeutics and preventatives for critical health conditions, including cancer. Antiproliferative effects are commonly ascribed to such compounds but ambiguity exists as to the principal mechanism of action and the universal benefit of exposure, particularly at high concentrations. Here, we identify heterogeneous responses within HL-60 promyelocytic leukaemia cells that explain contradictions in the reported origin of the antiproliferative action of kaempferol, a dietary abundant flavonoid. At > or =10 microM, kaempferol exposure is predominantly characterised by cell cycle alterations, notably a significant increase in S-phase and a progressive accumulation in G2-M with 10 and > or =20 microM kaempferol, respectively. However, a limited but consistent membrane damage is observed across the 1-100 microM exposure and at 1 microM occurs devoid from indices of apoptosis which are only consistently observed with > or =10 microM kaempferol treatment. At the most cytotoxic exposures, multiparametric flow cytometric analysis revealed distinct sub populations of cells. Cells with decreased size, typical of apoptosis and necrosis, possessed heightened caspase-3 activity, decreased anti-apoptotic Bcl-2 expression and changes to membrane asymmetry and integrity. The remaining population had elevated active caspase-3 but no change or a moderate increase in Bcl-2 expression and no plasma membrane alterations. Differentiation was not a significant factor in HL-60 growth inhibition. In conclusion, kaempferol-induced growth inhibition is dominated by cell cycle changes but involves a limited cytotoxicity, which we propose results from a membrane damage centred as well as an apoptotic process. This heterogeneity of response may confound the disease-preventative role and pharmacological application of this flavonoid.

Biphasic effect of falcarinol on caco-2 cell proliferation, DNA damage, and apoptosis.

The polyacetylene falcarinol, isolated from carrots, has been shown to be protective against chemically induced colon cancer development in rats, but the mechanisms are not fully understood. In this study CaCo-2 cells were exposed to falcarinol (0.5-100 microM) and the effects on proliferation, DNA damage, and apoptosis investigated. Low-dose falcarinol exposure (0.5-10 microM) decreased expression of the apoptosis indicator caspase-3 concomitantly with decreased basal DNA strand breakage. Cell proliferation was increased (1-10 microM), whereas cellular attachment was unaffected by <10 microM falcarinol. At concentrations above 20 microM falcarinol, proliferation of CaCo-2 cells decreased and the number of cells expressing active caspase-3 increased simultaneously with increased cell detachment. Furthermore, DNA single-strand breakage was significantly increased at concentrations above 10 microM falcarinol. Thus, the effects of falcarinol on CaCo-2 cells appear to be biphasic, inducing pro-proliferative and apoptotic characteristics at low and high concentrations of falcarinol, respectively.

The effect of short-term kaempferol exposure on reactive oxygen levels and integrity of human (HL-60) leukaemic cells.

Flavonoids may be a principal contributor to the cancer preventative activity of fruit- and vegetable-rich diets and there is interest in their use as dietary supplements. However, there is potential conflict between the cytoprotective and cytotoxic activities of flavonoids, and their efficacy as anti-cancer agents is unresolved. Here, the integrity and survival of HL-60 promyelocytic leukaemia cells following short-term (90 min) exposure to the dietary abundant flavonoid kaempferol (1-100 microM) is reported. Supplementation initially decreased reactive oxygen levels but, paradoxically, a dose-dependent increase in single-strand DNA breakage occurred. However, there was no increase in oxidised DNA purines or membrane damage. Following a 24-h recovery period in non-kaempferol supplemented media, DNA single-strand breakage had declined and kaempferol exposed and control cultures possessed similar reactive oxygen levels. A reduction in (3)H-thymidine incorporation occurred with > or =10 microM kaempferol. One hundred micromolar kaempefrol increased the proportion of cells in G(2)-M phase, the proportion of cells with a sub-G(1) DNA content and enhanced 'active' caspase-3 expression but only induced a loss of mitochondrial membrane potential within a minority of cells. The relevance of induced DNA damage within a non-overtly oxidatively stressed environment to the disease preventative and therapeutic use of kaempferol is discussed.

Increased salicylate concentrations in urine of human volunteers after consumption of cranberry juice.

The aim of this study was to assess whether regular consumption of cranberry juice results in elevations in urinary salicylate concentrations in persons not taking salicylate drugs. Two groups of healthy female subjects (11/group) matched for age, weight, and height consumed 250 mL of either cranberry juice or a placebo solution three times a day (i.e., 750 mL/day) for 2 weeks. At weekly intervals, salicylic acid and salicyluric acid (the major urinary metabolite of salicylic acid) concentrations were determined in urine by HPLC with electrochemical detection. Concentrations of salicylic acid in plasma were also determined. Consumption of cranberry juice was associated with a marked increase (p < 0.001) of salicyluric and salicylic acids in urine within 1 week of the intervention. After 2 weeks, there was also a small but significant (p < 0.05) increase in salicylic acid in plasma. The regular consumption of cranberry juice results in the increased absorption of salicylic acid, an anti-inflammatory compound that may benefit health.

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice.

Low B vitamin status is linked with human vascular disease. We employed a proteomic and biochemical approach to determine whether nutritional folate deficiency and/or hyperhomocysteinemia altered metabolic processes linked with atherosclerosis in ApoE null mice. Animals were fed either a control fat (C; 4 % w/w lard) or a high-fat [HF; 21 % w/w lard and cholesterol (0/15 % w/w)] diet with different B vitamin compositions for 16 weeks. Aorta tissue was prepared and global protein expression, B vitamin, homocysteine and lipoprotein status measured. Changes in the expression of aorta proteins were detected in response to multiple B vitamin deficiency combined with a high-fat diet (P < 0.05) and were strongly linked with lipoprotein concentrations measured directly in the aorta adventitia (P < 0.001). Pathway analysis revealed treatment effects in the aorta-related primarily to cytoskeletal organisation, smooth muscle cell adhesion and invasiveness (e.g., fibrinogen, moesin, transgelin, vimentin). Combined B vitamin deficiency induced striking quantitative changes in the expression of aorta proteins in atherosclerotic ApoE null mice. Deregulated expression of these proteins is associated with human atherosclerosis. Cellular pathways altered by B vitamin status included cytoskeletal organisation, cell differentiation and migration, oxidative stress and chronic inflammation. These findings provide new insight into the molecular mechanisms through which B vitamin deficiency may accelerate atherosclerosis.