Ochratoxin A-induced cytotoxicity in liver (HepG2) cells: Impact of serum concentration, dietary antioxidants and glutathione-modulating compounds

Abbrevations: BSO, buthionine sulfoximine; CAT, catechin; DMSO, dimethyl sulfoxide; DTNB, dithio-bis-nitrobenzoic acid; EGCG, epigallocatechin gallate; FCS, foetal calf serum; GSH, glutathione; IARC, international agency for research on cancer; NAC, N-acetylcysteine; NO, nitric oxide; NR, neutral red; OATP, organic anion-transporting polypeptide; OTA, ochratoxin A; PBS, phosphate buffered saline; QUE, quercetin; ROS, reactive oxygen species; ROSAC, rosmarinic acid; RPMI, roswell park memorial institute; α-TOC, α-tocopherol; α-TOC-P, α-tocopherol phosphat

Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization—Studies in gene-targeted replacement mice

Scope Of the three human apolipoprotein E (APOE) alleles, the ε3 allele is most common, which may be a result of adaptive evolution. In this study, we investigated whether the APOE genotype affects body weight and energy metabolism through regulation of fatty acid utilization. Methods and results Targeted replacement mice expressing the human APOE3 were significantly heavier on low- and high-fat diets compared to APOE4 mice. Particularly on high-fat feeding, food intake and dietary energy yields as well as fat mass were increased in APOE3 mice. Fatty acid mobilization determined as activation of adipose tissue lipase and fasting plasma nonesterified fatty acid levels were significantly lower in APOE3 than APOE4 mice. APOE4 mice, in contrast, exhibited higher expression of proteins involved in fatty acid oxidation in skeletal muscle. Conclusion Our data suggest that APOE3 is associated with the potential to more efficiently harvest dietary energy and to deposit fat in adipose tissue, while APOE4 carriers tend to increase fatty acid mobilization and utilization as fuel substrates especially under high-fat intake. The different handling of dietary energy may have contributed to the evolution and worldwide distribution of the ε3 allele

Pflanzen für die Gesundheit-Vorstellung eines neuen interdisziplinären Forschungsprojektes zum ökologischen Anbau von Arzneipflanzen

Ecologically grown medicinal plants containing bioactive compounds hold great poten-tial as high-value niche crops for farmers. However, the way to grow these plants differs from traditional crops. Growing techniques, harvest methods and postharvest handling of the raw material plays a crucial role regarding the quality of the raw material that the farmers can offer. The purpose of a new research project financed by EU-Interreg IIIA programme is among other things to carry out production, harvest and processing experiments with plants containing bioactive plant compounds that hold a preventive effect toward diabetes II. One of the project´s goals is to draw up cultivation instructions for the primary producers to use when cultivating the plants in question. Examples of the plants that are being examined in the project are Goat´s Rue (Galega officinalis) and Fenugreek (Trigonella foenum-graecum)

augustin

Green tea catechins (GTC) have been shown to inhibit the activities of enzymes involved in folate uptake. Hence, regular green tea drinkers may be at risk of impaired folate status. The present experiments aimed at studying the impact of dietary GTC on folate concentrations and metabolism. In a human pilot study (parallel design) healthy men consumed for 3 weeks 6 capsules (~670 mg GTC) per day (2 capsules with each principal meal) containing aqueous extracts of the leaves of Camellia sinensis (n=17) or placebo (n=16). No differences in plasma folate concentrations were observed between treatments. We further fed groups of 10 male rats diets fortified with 0, 0.05, 0.5, 1, or 5 g GTC/kg for 6 weeks. Only at the highest intake, GTC significantly decreased serum 5-methyl-tetrahydrofolate concentrations in rats, while mRNA concentrations of reduced folate carrier, proton-coupled folate transporter/heme carrier protein 1, and dihydrofolate reductase (DHFR) remained unchanged in intestinal mucosa. Using an in vitro enzyme activity assay, we observed a time-and dose-dependent inhibition of DHFR activity by epigallocatechin gallate and a green tea extract. Our data suggest that regular green tea consumption is unlikely to impair folate status in healthy males, despite the DHFR inhibitory activity of GTC. K e y w o r d s : folates, catechins, bioavailability, human, rat MATERIAL AND METHODS Dihydrofolate reductase activity The inhibition of human dihydrofolate reductase (DHFR) activity by (-) epigallocatechin gallate (EGCG) and a standardized green tea extract (Polyphenon 60 (P60); Sigma Chemical Co., St Louis, MO, USA) was measured using a commercial dihydrofolate reductase assay kit (Sigma-Aldrich) according to the manufacturer's protocol. Methotrexate, a well-known competitive DHFR inhibitor was used as a positive control. EGCG and P60 were dissolved in ultra pure-water (containing 1% ascorbic acid (w/v) (Merck KGaA, Darmstadt, Germany) to stabilize the catechins) on the day of the experiments. DHFR was used at a final activity of 1.5 x 10 -3 units per reaction. Final concentrations of EGCG and methotrexate were 1000, 100 and 10 nmol/L per reaction. P60 was used at final concentrations of 1428 .57, 142.86, and 14.29 µg/L and, thus, contained 1060 , 106, and 10.6 nmol/L EGCG and 1427.3 nmol/L of the gallated catechins (EGCG, ECG and gallocatechin gallate), respectively. Rat study Fifty male Wistar rats (Harlan Winkelmann GmbH, Borchen, Germany) with an initial body weight of 99.8 ± 2.0 g (mean ± SEM) were randomized into 5 groups of 10 animals each and housed pair-wise with sawdust bedding under controlled environmental conditions (23 ± 2°C and 65 ± 5% relative humidity, 12 h dark-light cycle). The rats were kept for 5 days on a folate-adjusted rat diet for growing animals containing 2 mg of folic acid/kg (C1027; Altromin GmbH, Lage, Germany) and thereafter received their respective experimental diets consisting of the standard diet supplemented with 0, 0.05, 0.5, 1, or 5 g green tea catechins per kg diet using P60 as the source of catechins (see The animal experiment was conducted in accordance with the German Guidelines and Regulations on Animal Care (Deutsches Tierschutzgesetz, 2006) and was approved by the University of Kiel Ethics Committee on Animal Care. Human pilot study Healthy males were recruited by advertisement at the University and local community of Reading (United Kingdom) and amongst volunteers who previously participated in nutritional trials at the Hugh Sinclair Human Nutrition Unit. Inclusion criteria were: male gender, 18-55 y of age, and a BMI in the range of 22-32 kg/m 2 . Subjects were excluded from the trial if they were diagnosed with any illness or on long-term medication, used dietary supplements, participated in >5 h of aerobic exercise activity per week, or were involved in a clinical trial within 3 months prior to the study. The study protocol was approved by the University of Reading ethics committee and all subjects gave written informed consent before participation. A standardized aqueous green tea extract prepared from the leaves of Camellia sinensis L. (a kind gift of Cognis Deutschland GmbH & Co KG, Monheim am Rhein, Germany) was used to make the green tea extract (GTE) capsules. The composition of the GTE is given in The trial was designed as a double-blind placebo-controlled parallel study. Thirty-one volunteers were randomly assigned to one of two treatment groups (GTE, n=16 or placebo, n=15) with similar BMI and age (data not shown). Subjects took 6 capsules per day, two with each principal meal, for 3 weeks and were instructed to limit their daily tea and coffee consumption to ≤ 3 cups, but to otherwise maintain their normal diet and exercise patterns. Compliance was determined by counting of the returned capsules at the end of the trial and was high (>98%). Blood samples (20 ml) were drawn into tubes containing 0.05 mL 15% K 3 EDTA (Vacutainer; Becton Dickinson UK Ltd., Oxford, UK) after an overnight fast on the first and last day of the intervention period. Plasma was immediately obtained by centrifugation (1,000 x g, 10 min) and 3 ml aliquots were stored at -80°C until analysis. Folate quantification by HPLC Procedures for extraction and purification of folates from human plasma and rat serum and liver samples by strong anion exchange solid-phase extraction were described previously by Witthoft et al. (18). Dialysed rat serum (500 µl/g) was used to ensure complete deconjugation of folate polyglutamates in liver samples; modified from Patring et al. (19). Analyses were performed using an HPLC system (Agilent 1100) consisting of a 104 10-formyltetrahydrofolate (10-HCO-H 4 folate), and 5,10-methenyltetrahydrofolate (5,10-CH + -H 4 folate) (a gift of Merck Eprova AG, Schaffhausen, Switzerland, except 10-HCO-H 4 folate, which was purchased from Schircks Laboratories, Jona, Switzerland). Quantification was based on a multilevel (n=7) external calibration curve with a linear range over 1.2-118.0 ng/mL for H 4 folate, 0.6-93.1 ng/mL for 5-CH 3 -H 4 folate, 0.9-184.1 ng/mL for 10-HCO-H 4 folate and 9.3-184.5 ng/mL for 5,10-CH + -H 4 folate. mRNA quantification RNA was isolated from rat duodenal mucosa using the RNeasy Lipid Tissue Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. DNA digestion was performed with RNase-Free DNase Set (Qiagen). RNA integrity was checked by electrophoresis on a denaturing agarose gel and ethidium bromide staining. The concentration and purity of isolated RNA was determined by measuring the absorbance (AB) at 260 and 280 nm in a spectrophotometer (DU800, Beckmann Instruments; Munich, Germany). A ratio of >1.8 between AB 260nm and AB 280nm was considered as acceptable. RNA aliquots were stored at -80°C until analysis. Primer pairs of β-actin, reduced folate carrier (RFC) and proton-coupled folate transporter/heme carrier protein-1 (PCFT/HCP1) were designed to the corresponding sequences of Rattus norvegicus mRNA with Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/ primer3_www.cgi; 03.05.2007) and purchased from MWGBiotech AG (Ebersberg, Germany). The sequences of primers used in this study were as follows: Sense primer for β-actin, 5´-GGGGTGTTGAAGGTCTCAAA-3´, antisense primer for β-actin, 5´-TGTCACCAACTGGGACGATA-3´; sense primer for RFC, 5´-GGCTCGTGTTCTACCTCTGC-3´, antisense primer for RFC, 5´-GGTAGTCGGTGAGCAGGAAG-3´; sense primer for PCFT/HCP1, 5´-TGAGCTAAGCACACCCCTCT-3´, antisense primer for PCFT/HCP1, 5´-TCCGTACCCTGTGAACATGA-3´. The product size was 90 base pair (bp) for β-actin; 183 bp for RFC and 217 bp for PCFT/HCP1. QuantiTect ® Primer Assay (Qiagen) was used for DHFR mRNA amplification, with a product size of 88 bp. For one-step quantitative reverse transcriptase polymerase chain reaction (one-step qRT-PCR) two aliquots of RNA were amplified. External relative standard curves of total RNA were determined with each run. Data was normalized by dividing the concentrations of RFC, PCFT/HCP1 or DHFR by the concentrations of β-actin mRNA. Each PCR reaction (final volume 20 µl) contained 0.5 µmol/L of each primer, 10 µl of 2x QuantiTect ® SYBR ® Green RT-PCR Master Mix (Qiagen), 0.2 µl QuantiTect RT-Mix (Qiagen), 8 µl of RNA dilution and 1.4 µl water. Real-time cycler conditions were set according to the manufacturers protocol to 40 cycles with annealing temperatures of 56°C for β-actin, 59°C for RFC, 56°C for PCFT/HCP1 and 55°C for DHFR, respectively. Quantification and melting curves of the amplified products were analysed using the RotorGene 6.0 software (Corbett Lifescience; Sydney, Australia). Melting curve analyses and agarose gel electrophoresis with ethidium bromide staining were performed to exclude non-specific products. Statistical analyses Statistical calculations were performed with GraphPad Prism 4 software (GraphPad Software Inc., San Diego, CA, USA). Analyses of the data from the rat study and the in vitro assay were performed by means of a one-way ANOVA followed by Dunnetts test for multiple comparisons of group means between animals receiving GTC or control diet. Analyses of the data from the human pilot study were performed by means of a paired Student's t-test for comparison of baseline vs. treatment and by means of an unpaired Student's t-test for comparisons between subjects receiving GTE or placebo. Reported values are means ± SEM and effects were considered significant at P<0.05. RESULTS Dihydrofolate reductase activity in vitro Both pure EGCG and P60, at concentrations of 1000 for EGCG and 1060 nmol/L for EGCG from P60, respectively, time-dependently inhibited DHFR activity Serum and liver folate concentrations in rats Feed consumption and final body mass (318.7 ± 4.8 g) of the Wistar rats were similar in all groups. Intake of diets containing 0.5% GTC over a period of 42 days significantly decreased the serum concentration of 5-CH 3 -H 4 folate compared to control rats, whereas the concentrations of H 4 folate remained unchanged ( Relative mRNA levels of reduced folate carrier and dihydrofolate reductase in rat duodenal mucosa The housekeeping gene β-actin was expressed at similar levels in all animals and no significant differences in the relative mRNA levels of RFC, PCFT/HCP1 or DHFR in the duodenal mucosa were observed Plasma folate concentrations in humans Consumption of 670 mg of GTC per day or placebo did not affect plasma folate concentrations in healthy male volunteers. No significant differences in plasma concentrations of 5-CH 3 -H 4 folate were observed between the treatment groups at baseline (placebo, 16.3 ± 2.6 nmol/L; GTE, 19.1 ± 2.4 nmol/L) or after intervention (placebo, 15.5 ± 2.1 nmol/L; GTE, 17.6 ± 2.4 nmol/L). DISCUSSION Green tea is a widely consumed beverage in many countries and contains appreciable amounts of polyphenols. Catechins (flavanols) are the major subclass of bioactive compounds within the polyphenol fraction of green tea. Epidemiological studies associated a high dietary intake of catechins with a reduced risk to suffer from a variety of diseases (reviewed in 20), including certain forms of cancer (21). The underlying molecular and cellular mechanisms by which green tea catechins may mediate anticarcinogenic acitivty seem to be diverse: Cell culture experiments as well as studies in rodents indicate that green tea catechin may inhibit angiogenesis via a down-regulation of vascular endothelial growth factor (reviewed in 22). Furthermore it has been suggested that the anticancer activity of green tea catechins against different kind of cancers may find an explanation in direct targeting of lipid rafts (23). Recent in vitro studies have shown that epigallocatechin gallate (EGCG), the predominant catechin in green tea, competitively inhibits the enzyme dihydrofolate reductase (DHFR) (9, 13). DHFR inhibition is the mechanism by which so-called antifolates, such as the cytostatic drug methotrexate, inhibit cell division and reduce tumor growth (15, 24). Co-administration of folic acid and the DHFR inhibitors methotrexate and pyrimethamine, respectively, reduced plasma folate concentrations in rats The commercial green tea extract Polyphenon 60 (P60) used in the rat study and its principle bioactive ingredient EGCG inhibited DHRF activity time-and concentration-dependently in vitro In order to study whether or not the effects observed in vitro bear a meaning for the more complex physiological processes in vivo, Wistar rats were fed for 42 days with diets fortified with increasing concentrations of green tea catechins (GTC) using a standardized green tea extract (P60). The diets contained 2 mg folic acid per kg, which is equivalent to twice the dietary recommendations for laboratory rats as given by the National Research Council (28). It is noteworthy that folates synthesized by the microflora of the large intestine are absorbed and may significantly contribute to blood folate concentrations (reviewed in 29). The diet used in this study was therefore formulated to provide a minimum of substrate to the intestinal microflora to limit bacterial folate synthesis. Only in those animals fed the highest concentrations of the green tea extract (0.5% GTC), did we observe a significant decrease in serum 5-CH 3 -H 4 folate concentrations as compared to the control group ( At a given substrate affinity and substrate concentration, the capacity of enzymatic turnover of folates as well as the amount of their carrier-mediated transport across cellular membranes is mainly affected by the amount of enzymes/carriers present at the tissue level. Because catechins are known to alter the gene expression for a variety of proteins (35), we quantified relative mRNA concentrations of the RFC, PCFT/HCP1, and DHFR in the duodenal mucosa of rats fed GTC. No significant differences in mRNA concentrations of RFC, PCFT/HCP1, and of DHFR were found between the experimental groups The current findings suggested that GTC might decrease serum folate concentrations only if supplied at supra-nutritional doses. A 70 kg human would have to drink almost 100 cups of green tea infusion per day to match the highest dose fed to rats in the present study. Because such a human study would be unfeasible as well as unrealistic, we designed a pilot study with a standardized green tea extract to assess whether or not regular consumption of high doses of GTC might affect plasma folate concentrations in humans. The intake of 670 mg of GTC per day, which corresponds to about 20 cups of green tea, caused no significant differences in plasma concentrations of 5-CH 3 -H 4 folate between the treatment and placebo groups, both of which consuming a normal diet containing on average ~328 ± 26 µg folate/d. Insufficient dietary intake of folates for as short as 2-3 weeks has been reported to result in reduced blood concentrations of the vitamin (30). Our findings therefore suggest that green tea drinking is unlikely to affect plasma folate concentrations in healthy, free-living subjects and that a longer treatment period and/or even higher doses of dietary GTC may be necessary to induce changes in folate concentrations, if possible at all. Further human studies with GTC and a standardized supply of folic acid (in the absence of naturally occurring reduced folates) are warranted to investigate the influence of GTC on DHFR activity in vivo. In addition, the measurement of (oxidized) serum folic acid should be considered because folic acid has been found in serum of subjects consuming folic acid-fortified foods for 5 d (11). Based on the experiments presented here, it appears unlikely that daily green tea consumption, even at high levels, may affect folate concentrations in healthy humans. Acknowledgement

Association between genetic variants in the Coenzyme Q10 metabolism and Coenzyme Q10 status in humans

<p>Abstract</p> <p>Background</p> <p>Coenzyme Q₁₀(CoQ₁₀) is essential for mitochondrial energy production and serves as an antioxidants in extra mitochondrial membranes. The genetics of primary CoQ₁₀deficiency has been described in several studies, whereas the influence of common genetic variants on CoQ₁₀status is largely unknown. Here we tested for non-synonymous single-nucleotidepolymorphisms (SNP) in genes involved in the biosynthesis (CoQ3^G272S, CoQ6^M406V, CoQ7^M103T), reduction (NQO1^P187S, NQO2^L47F) and metabolism (apoE3/4) of CoQ₁₀and their association with CoQ₁₀status. For this purpose, CoQ₁₀serum levels of 54 healthy male volunteers were determined before (T₀) and after a 14 days supplementation (T₁₄) with 150 mg/d of the reduced form of CoQ₁₀.</p> <p>Findings</p> <p>At T₀, the CoQ₁₀level of heterozygous NQO1^P187Scarriers were significantly lower than homozygous S/S carriers (0.93 ± 0.25 μM versus 1.34 ± 0.42 μM, p = 0.044). For this polymorphism a structure homology-based method (PolyPhen) revealed a possibly damaging effect on NQO1 protein activity. Furthermore, CoQ₁₀plasma levels were significantly increased in apoE4/E4 genotype after supplementation in comparison to apoE2/E3 genotype (5.93 ± 0.151 μM versus 4.38 ± 0.792 μM, p = 0.034). Likewise heterozygous CoQ3^G272Scarriers had higher CoQ₁₀plasma levels at T₁₄compared to G/G carriers but this difference did not reach significance (5.30 ± 0.96 μM versus 4.42 ± 1.67 μM, p = 0.082).</p> <p>Conclusions</p> <p>In conclusion, our pilot study provides evidence that NQO1^P187Sand apoE polymorphisms influence CoQ₁₀status in humans.</p

Living long and well: prospects for a personalized approach to the medicine of ageing

Research into ageing and its underlying molecular basis enables us to develop and implement targeted interventions to ameliorate or cure its consequences. However, the efficacy of interventions often differs widely between individuals, suggesting that populations should be stratified or even individualized. Large-scale cohort studies in humans, similar systematic studies in model organisms as well as detailed investigations into the biology of ageing can provide individual validated biomarkers and mechanisms, leading to recommendations for targeted interventions. Human cohort studies are already ongoing, and they can be supplemented by in silico simulations. Systematic studies in animal models are made possible by the use of inbred strains or genetic reference populations of mice. Combining the two, a comprehensive picture of the various determinants of ageing and ‘health span' can be studied in detail, and an appreciation of the relevance of results from model organisms to humans is emerging. The interactions between genotype and environment, particularly the psychosocial environment, are poorly studied in both humans and model organisms, presenting serious challenges to any approach to a personalized medicine of ageing. To increase the success of preventive interventions, we argue that there is a pressing need for an individualized evaluation of interventions such as physical exercise, nutrition, nutraceuticals and calorie restriction mimetics as well as psychosocial and environmental factors, separately and in combination. The expected extension of the health span enables us to refocus health care spending on individual prevention, starting in late adulthood, and on the brief period of morbidity at very old ag

Robot-assisted surgery for the management of apical prolapse: a bicentre prospective cohort study

Objective: Robot‐assisted surgery is a recognized treatment for pelvic‐organ prolapse. Many of the surgical subgroup outcomes for apical prolapse are reported together leading to a paucity of homogenous data. Design: Prospective observational cohort study (https://clinicaltrials.gov; identifier NCT01598467) assessing outcomes for homogeneous subgroups of robot‐assisted apical prolapse surgery. Setting: Two European tertiary referral hospitals. Population: Consecutive patients undergoing robot‐assisted sacrocolpopexy (RASC) and supracervical hysterectomy with sacrocervicopexy (RSHS). Methods: Anatomical cure (simplified Pelvic Organ Prolapse Quantification (sPOPQ) stage 1,), subjective cure (symptoms of bulge) and quality of life (Pelvic Floor Impact Questionnaire [PFIQ‐7]). Main Outcome measures: Primary outcome: anatomical and subjective cure. Secondary outcomes: surgical safety and intraoperative variables. Results: Total 305 patients included (RASC N=188, RSHS N=117). Twelve months follow‐up available for 144 (RASC 76.6%) and 109 (RSHS 93.2%). Anatomical success of the apical compartment occurred in 91% (RASC) and in 99% (RSHS). In all compartments, success percentages were 67% and 65% respectively. Most recurrences were anterior compartment (15.7% RASC [symptomatic 12.1%]; 22.9% RSHS [symptomatic 4.8%]). Symptoms of bulge improved from 97.4% to 17.4% (p<0.0005). PFIQ‐7 scores improved from 76.7 ± 62.3 to 13.5 ± 31.1 (p<0.0005). Duration of surgery increased significantly in RSHS (183.1 ± 38.2 versus 145.3 ± 29.8 [p<0.0005]). Intraoperative complications and conversion rates were low (RASC: 5.3% and 4.3%; RSHS: 0.0% and 0.0%). Four severe postoperative complications occurred after RASC (2.1%) and one after RSHS (1.6%). Conclusion: This is the largest reported prospective cohort study on robot‐assisted apical prolapse surgery. Both procedures are safe, with durable results

Total energy expenditure is repeatable in adults but not associated with short-term changes in body composition

Low total energy expenditure (TEE, MJ/d) has been a hypothesized risk factor for weight gain, but repeatability of TEE, a critical variable in longitudinal studies of energy balance, is understudied. We examine repeated doubly labeled water (DLW) measurements of TEE in 348 adults and 47 children from the IAEA DLW Database (mean ± SD time interval: 1.9 ± 2.9 y) to assess repeatability of TEE, and to examine if TEE adjusted for age, sex, fat-free mass, and fat mass is associated with changes in weight or body composition. Here, we report that repeatability of TEE is high for adults, but not children. Bivariate Bayesian mixed models show no among or within-individual correlation between body composition (fat mass or percentage) and unadjusted TEE in adults. For adults aged 20-60 y (N = 267; time interval: 7.4 ± 12.2 weeks), increases in adjusted TEE are associated with weight gain but not with changes in body composition; results are similar for subjects with intervals >4 weeks (N = 53; 29.1 ± 12.8 weeks). This suggests low TEE is not a risk factor for, and high TEE is not protective against, weight or body fat gain over the time intervals tested