Short-Term Dietary Intervention with Cooked but Not Raw Brassica Leafy Vegetables Increases Telomerase Activity in CD8+ Lymphocytes in a Randomized Human Trial.

Telomerase in T lymphocytes is dynamic and limited evidence from epidemiological studies indicates that the enzyme can be modulated in peripheral lymphocytes by dietary and lifestyle factors. The differential effect of dietary intervention on T cell subsets has not been investigated so far. Brassica vegetables are known for their multiple beneficial effects on human health, and here, the effect of a five-day short-term intervention with raw or cooked leaves of Brassica carinata on telomerase activity in CD4+ and CD8+ T cells from 22 healthy volunteers was investigated in a randomized single-blind, controlled crossover study. Blood samples were collected before and after intervention, and CD4+/CD8+ T lymphocytes were isolated. Telomerase activity was quantified using the TRAP-ELISA assay. Intervention with both preparations led to a marginal increase in telomerase activity of CD4+ cells compared to the baseline level. In CD8+ cells, a significant increase in telomerase activity (25%, p < 0.05) was seen after intervention with the cooked material. An increase in telomerase activity in CD8+ cells of healthy volunteers could be regarded as beneficial in terms of helping with the cell-mediated immune response. Whether a Brassica intervention has long-term effects on telomere extension in specific T cell subsets needs to be determined.

Are Raw Brassica Vegetables Healthier Than Cooked Ones? A Randomized, Controlled Crossover Intervention Trial on the Health-Promoting Potential of Ethiopian Kale.

The present human intervention trial investigated the health-promoting potential of B. carinata, with a focus on effects of thermal processing on bioactivity. Twenty-two healthy subjects consumed a B. carinata preparation from raw (allyl isothiocyanate-containing) or cooked (no allyl isothiocyanate) leaves for five days in a randomized crossover design. Peripheral blood mononuclear cells were exposed to aflatoxin B1 (AFB1), with or without metabolic activation using human S9 mix, and subsequently analyzed for DNA damage using the comet assay. Plasma was analyzed for total antioxidant capacity and prostaglandin E2 (PGE2) levels. Cooked B. carinata significantly reduced DNA damage induced by AFB1 as compared to baseline levels (+S9 mix: 35%, -S9 mix: 33%, p ≤ 0.01, respectively). Raw B. carinata only reduced DNA damage by S9-activated AFB1 by 21% (p = 0.08). PGE2 plasma levels were significantly reduced in subjects after consuming raw B. carinata. No changes in plasma antioxidant capacity were detectable. A balanced diet, including raw and cooked Brassica vegetables, might be suited to fully exploit the health-promoting potential. These results also advocate the promotion of B. carinata cultivation in Eastern Africa as a measure to combat effects of unavoidable aflatoxin exposure.

African Nightshade (Solanum scabrum Mill.): Impact of Cultivation and Plant Processing on Its Health Promoting Potential as Determined in a Human Liver Cell Model.

Plant cultivation and processing may impact nutrient and phytochemical content of vegetables. The present study aimed at determining the influence of cultivation and processing on the health promoting capacity of African nightshade (Solanum scabrum Mill.) leaves, an indigenous vegetable, rich in nutrients and phytochemicals. Anti-genotoxicity against the human liver carcinogen aflatoxin B1 (AFB1) as determined by the comet assay and radical oxygen species (ROS) scavenging capacity of ethanolic and aqueous extracts were investigated in human derived liver (HepG2) cells. ROS scavenging activity was assessed using electron paramagnetic spin resonance and quantification of ARE/Nrf2 mediated gene expression. The cultivation was done under different environmental conditions. The processing included fermentation and cooking; postharvest ultraviolet irradiation (UV-C) treatment was also investigated. Overall, S. scabrum extracts showed strong health promoting potential, the highest potential was observed with the fermented extract, which showed a 60% reduction of AFB1 induced DNA damage and a 38% reduction in FeSO4 induced oxidative stress. The content of total polyphenols, carotenoids and chlorophylls was indeed affected by cultivation and processing. Based on the present in vitro findings consumption of S. scabrum leaves could be further encouraged, preferentially after cooking or fermentation of the plant.

Low-dose levels of bisphenol A inhibit telomerase via ER/GPR30-ERK signalling, impair DNA integrity and reduce cell proliferation in primary PBMC.

Controversy exists about the human health risk of environmental exposure to bisphenol A (BPA). Telomerase activity is emerging both as biomarker and contributing factor for age-related diseases. The effects of BPA exposure at 1-1000 nM on telomerase, DNA integrity and cell proliferation were investigated in PBMC from human donors. Telomerase activity was determined by TRAP-ELISA assay and mRNA expression by qRT-PCR. Mechanistic studies were carried out on the ER/GPR30-ERK pathway using specific inhibitors/antagonists, the comet assay to quantify DNA damage and flow cytometry for cell proliferation. 24 h BPA exposure inhibited telomerase in a non-monotonic pattern with a peak inhibition of 32% at 1 nM (p ≤ 0.01). A significant telomerase inhibition was evident at 1 h after exposure with a minimum at 6 h. Elevated levels of DNA damage frequency and decrease in cell proliferation were evident upon long-term exposure. The results further demonstrate that BPA triggered rapidly an ER/GPR30-ERK transduction pathway that leads to decreased telomerase activity in human PBMC. This is the first study to demonstrate adverse impact of BPA at levels of current human exposure on telomerase in normal cells, mediated by ER/GPR30-ERK. The results suggest a potentially harmful influence of BPA on immune cells and should be addressed in future studies.

In vitro observations and in silico predictions of xenoestrogen mixture effects in T47D-based receptor transactivation and proliferation assays.

Within endocrine disruptor research, evaluation and interpretation of mixture effects and the predictive value for downstream responses still warrant more in-depth investigations. We used an estrogen receptor (ER)-mediated reporter gene assay (ER-CALUX®) and a cell proliferation assay (WST-1 assay), both based on the T47D breast cancer cell line, to test mixtures of heterogeneous xenoestrogens. Observed concentration-response curves were compared to those predicted by the concepts of concentration addition (CA), generalized concentration addition (GCA), and a novel full logistic model (FLM). CA performed better regarding mixture potency (EC50 values), whereas GCA was superior in predicting mixture efficacy (maximal response). In comparison, FLM proved to be highly suitable for in silico mixture effect prediction, combining advantages of both CA and GCA. The inter-assay comparison revealed that ER activation is not necessarily predictive for induction of cell proliferation. The results support the use of models like CA, GCA, or FLM in mixture effect evaluation. However, we conclude that reliable estimations regarding the disruptive potential of mixtures of endocrine active substances require an integrative approach considering more than one assay/endpoint to avoid misinterpretations. The formazan-based WST-1 proliferation assay might be a possible alternative to commonly used proliferation assays in endocrine disrupter research.

Normal human immune cells are sensitive to telomerase inhibition by Brassica-derived 3,3-diindolylmethane,partly mediated via ERα/ß-AP1 signaling.

SCOPE: Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) from Brassica plants are regarded as promising anticancer phytochemicals. The enzyme telomerase is a very attractive target for cancer therapeutics; in normal cells such as lymphocytes, it plays a decisive role for cell maintenance. The effect of I3C and DIM on telomerase in normal human immune cells (PBMC) was studied compared to leukaemia cells (HL-60). Signalling of telomerase regulation via estrogen receptor (ER) was addressed. METHODS AND RESULTS: Short-term treatment with I3C and DIM inhibited telomerase activity in leukaemia cells (>30 µM I3C; >3 µM DIM). In CD3/CD28 activated PBMC, inhibition was stronger, though (>3 µM I3C; >1 µM DIM). DIM long-term treatment resulted in DNA damage induction and proliferation inhibition in PBMC as determined by the comet assay and CFSE staining, respectively. A relevance of ERα/ß-AP1 signaling for telomerase inhibition on enzyme activity, but not transcription level became evident indicating a nonclassical mode for ER regulation of telomerase by DIM. CONCLUSION: Although desired in cancer cells, this study identified a potential adverse impact of I3C and DIM on telomerase action in normal human immune cells, partly mediated by an ER-dependent mechanism. These new findings should be considered for potential chronic high-dose chemoprevention strategies using these compounds.

The role of plant processing for the cancer preventive potential of Ethiopian kale (Brassica carinata).

Background: Ethiopian kale (Brassica carinata) is a horticulturally important crop used as leafy vegetable in large parts of East and Southern Africa. The leaves are reported to contain high concentrations of health-promoting secondary plant metabolites. However, scientific knowledge on their health benefits is scarce. Objective: This study aimed to determine the cancer preventive potential of B. carinata using a human liver in vitro model focusing on processing effects on the pattern of secondary plant metabolites and bioactivity. Design: B. carinata was cultivated under controlled conditions and differentially processed (raw, fermented, or cooked) after harvesting. Human liver cancer cells (HepG2) were treated with ethanolic extracts of raw or processed B. carinata leaves and analyzed for their anti-genotoxic, anti-oxidant, and cytostatic potential. Chemical analyses were carried out on glucosinolates including breakdown products, phenolic compounds, carotenoids, and chlorophyll content. Results: Pre-treatment with B. carinata extracts concentration dependently reduced aflatoxin-induced DNA damage in the Comet assay, reduced the production of reactive oxygen species as determined by electron paramagnetic resonance spectroscopy, and induced Nrf2-mediated gene expression. Increasing extract concentrations also promoted cytostasis. Processing had a significant effect on the content of secondary plant metabolites. However, different processing methodologies did not dramatically decrease bioactivity, but enhanced the protective effect in some of the endpoints studied. Conclusion: Our findings highlight the cancer preventive potential of B. carinata as indicated by the protection of human liver cells against aflatoxin in vitro. In general, consumption of B. carinata should be encouraged as part of chemopreventive measures to combat prevalence of aflatoxin-induced diseases.

Combined effects of dietary polyunsaturated fatty acids and parasite exposure on eicosanoid-related gene expression in an invertebrate model.

Eicosanoids derive from essential polyunsaturated fatty acids (PUFA) and play crucial roles in immunity, development, and reproduction. However, potential links between dietary PUFA supply and eicosanoid biosynthesis are poorly understood, especially in invertebrates. Using Daphnia magna and its bacterial parasite Pasteuria ramosa as model system, we studied the expression of genes coding for key enzymes in eicosanoid biosynthesis and of genes related to oogenesis in response to dietary arachidonic acid and eicosapentaenoic acid in parasite-exposed and non-exposed animals. Gene expression related to cyclooxygenase activity was especially responsive to the dietary PUFA supply and parasite challenge, indicating a role for prostanoid eicosanoids in immunity and reproduction. Vitellogenin gene expression was induced upon parasite exposure in all food treatments, suggesting infection-related interference with the host's reproductive system. Our findings highlight the potential of dietary PUFA to modulate the expression of key enzymes involved in eicosanoid biosynthesis and reproduction and thus underpin the idea that the dietary PUFA supply can influence invertebrate immune functions and host-parasite interactions.

The Brassica epithionitrile 1-cyano-2,3-epithiopropane triggers cell death in human liver cancer cells in vitro.

SCOPE: Glucosinolates are secondary metabolites present in Brassica vegetables. Alkenyl glucosinolates are enzymatically degraded forming nitriles or isothiocyanates, but in the presence of epithiospecifier protein, epithionitriles are released. However, studies on the occurrence of epithionitriles in Brassica food and knowledge about their biological effects are scarce. METHODS AND RESULTS: Epithionitrile formation from glucosinolates of seven Brassica vegetables was analyzed using GC-MS and HPLC-DAD. Bioactivity of synthetic and plant-derived 1-cyano-2,3-epithiopropane (CETP) - the predominant epithionitrile in Brassica vegetables - in three human hepatocellular carcinoma (HCC) cell lines and primary murine hepatocytes was also evaluated. The majority of the Brassica vegetables were producers of nitriles or epithionitriles as hydrolysis products and not of isothiocyanates. For example, Brussels sprouts and savoy cabbage contained up to 0.8 µmol CETP/g vegetable. Using formazan dye assays, concentrations of 380-1500 nM CETP were observed to inhibit the mitochondrial dehydrogenase activity of human HCC cells without impairment of cell growth. At 100-fold higher CETP concentrations, cell death was observed. Presence of plant matrix increased CETP-based toxicity. CONCLUSION: These in vitro data provide no indication that epithionitriles will severely affect human health by Brassica consumption. In contrast to isothiocyanates, no evidence of selective toxicity against HCC cells was found.

A dietary polyunsaturated fatty acid improves consumer performance during challenge with an opportunistic bacterial pathogen.

A dietary deficiency in polyunsaturated fatty acids (PUFAs) and/or sterols can severely constrain growth and reproduction of invertebrate consumers. Single nutrients are potentially assigned to different physiological processes, for example to support defence mechanisms; therefore, lipid requirements of healthy and pathogen-challenged consumers might differ. In an oral exposure experiment, we explored the effects of dietary PUFAs and cholesterol on growth, reproduction and survival of an aquatic key herbivore (Daphnia magna) exposed to an opportunistic pathogen (Pseudomonas sp.). We show that healthy and pathogen-challenged D. magna are strongly albeit differentially affected by the biochemical composition of their food sources. Supplementation of a C20 PUFA-deficient diet with arachidonic acid (ARA) resulted in increased survival and reproduction of pathogen-challenged D. magna. We propose that the observed benefit of consuming an ARA-rich diet during pathogen challenge is conveyed partially via ARA-derived eicosanoids. This study is one of the first to consider the importance of dietary PUFAs in modifying fitness parameters of pathogen-challenged invertebrate hosts. Our results suggest that dietary PUFA supply should receive increased attention in host-microorganisms interactions and invertebrate disease models to better understand and predict disease dynamics in natural populations.

Dietary supply with polyunsaturated fatty acids and resulting maternal effects influence host--parasite interactions.

BACKGROUND: Interactions between hosts and parasites can be substantially modulated by host nutrition. Polyunsaturated fatty acids (PUFAs) are essential dietary nutrients; they are indispensable as structural components of cell membranes and as precursors for eicosanoids, signalling molecules which act on reproduction and immunity. Here, we explored the potential of dietary PUFAs to affect the course of parasitic infections using a well-established invertebrate host - parasite system, the freshwater herbivore Daphnia magna and its bacterial parasite Pasteuria ramosa. RESULTS: Using natural food sources differing in their PUFA composition and by experimentally modifying the availability of dietary arachidonic acid (ARA) and eicosapentaenoic acid (EPA) we examined PUFA-mediated effects resulting from direct consumption as well as maternal effects on offspring of treated mothers. We found that both host and parasite were affected by food quality. Feeding on C20 PUFA-containing food sources resulted in higher offspring production of hosts and these effects were conveyed to a great extent to the next generation. While feeding on a diet containing high PUFA concentrations significantly reduced the likelihood of becoming infected, the infection success in the next generation increased whenever the maternal diet contained PUFAs. We suggest that this opposing effect was caused by a trade-off between reproduction and immunity in the second generation. CONCLUSIONS: Considering the direct and maternal effects of dietary PUFAs on host and parasite we propose that host - parasite interactions and thus disease dynamics under natural conditions are subject to the availability of dietary PUFAs.

The potential of dietary polyunsaturated fatty acids to modulate eicosanoid synthesis and reproduction in Daphnia magna: a gene expression approach.

Nutritional ecology of the aquatic model genus Daphnia has received much attention in past years in particular with regard to dietary polyunsaturated fatty acids (PUFAs) which are crucial for growth and reproduction. Besides their significant role as membrane components, C20 PUFAs serve as precursors for eicosanoids, hormone-like mediators of reproduction, immunity and ion transport physiology. In the present study we investigate transcriptomic changes in Daphnia magna in response to different algal food organisms substantially differing in their PUFA composition using quantitative real-time PCR and relate them to concomitantly documented life history data. The selection of target genes includes representatives that have previously been shown to be responsive to the eicosanoid biosynthesis inhibitor ibuprofen. The beneficial effect of C20 PUFA-rich food on reproduction and population growth rates was accompanied by an increased vitellogenin (DmagVtg1) gene expression in D. magna. Additionally, genes involved in eicosanoid signaling were particularly influenced by dietary C20 PUFA availability. For example, the cyclooxygenase gene (Cox), coding for a central enzyme in the eicosanoid pathway, was highly responsive to the food treatments. Our results suggest that dietary PUFAs are fundamental in D. magna physiology as substrate for eicosanoid synthesis and that these eicosanoids are important for D. magna reproduction.