Effect of lectins on the transport of food factors in caco-2 cell monolayers

The effect of three plant lectins, soybean lectin (SBA), Japanese jack bean lectin (CGA), and wheat germ lectin (WGA), on the transport of various food factors, such as isoflavones, quercetin, dipeptides, and calcium ions, were investigated by use of an intestinal tract model, Caco-2 cell monolayers. The lectins increased the isoflavone transport but had no effect on aglycon transport. SBA increased the transport of quercetin glycosides, whereas CGA and WGA had no effect. The lectins increased the transport of calcium ions but showed no effect on the transport of dipeptides, carnosine, and anserine. Although SBA did not change the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayers, CGA and WGA decreased the TER value. These results indicate that plant lectins affect the transport of food factors in different manners, presumably due to their specific sugar binding activity.     

Dual action of sulforaphane in the regulation of thioredoxin reductase and thioredoxin in human HepG2 and Caco-2 cells

We have previously demonstrated that sulforaphane is a potent inducer for thioredoxin reductase in HepG2 and MCF-7 cells (Zhang et al. Carcinogenesis 2003, 24, 497-503; Wang et al. J. Agric. Food Chem. 2005, 53, 1417-1421). In this study, we have shown that sulforaphane is not only an inducer for thioredoxin reductase but also an inducer for its substrate, thioredoxin in HepG2, and undifferentiated Caco-2 cells. Sulforaphane acts at two levels in the regulation of thioredoxin reductase/thioredoxin system by the upregulation of the expression of both the enzyme and the substrate. In human hepatoma HepG2 cells, sulforaphane induced thioredoxin reductase mRNA and protein by 4- and 2-fold, respectively, whereas thioredoxin mRNA was induced 2.9-fold and thioredoxin protein was unchanged in whole cell extracts, but an increase in nuclear accumulation (1.8-fold) was observed. Moreover, the induction of thioredoxin reductase was found faster than that of thioredoxin. The effects of PI3K and MAPK kinase inhibitors, LY294002, PD98059, SP600125, and SB202190, have been investigated on the sulforaphane-induced expression of thioredoxin reductase and thioredoxin. PD98059 abrogates the sulforaphane-induced thioredoxin reductase at both mRNA and protein levels in HepG2 cells, although other inhibitors were found less effective. However, both PD98059 and LY294002 significantly decrease thioredoxin mRNA expression in HepG2 cells. None of the inhibitors tested were able to modulate the level of expression of either thioredoxin reductase mRNA or protein in Caco-2 cells suggesting that there are cell-specific responses to sulforaphane. In summary, the dietary isothiocyanate, sulforaphane, is important in the regulation of thioredoxin reductase/thioredoxin redox system in cells.     

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.     

Peptides isolated from in vitro digests of milk enhance iron uptake by caco-2 cells

Milk proteins, during digestion, produce a range of biologically active peptides. Among those are peptides that may enhance iron absorption. The objective of this project was to investigate the effect of isolated milk peptides on iron uptake. Cow's milk, 0% fat, was subjected to a modified in vitro digestion process. The milk digest was further fractionated by gel filtration. All eluted fractions as well as beta-casein synthetic peptides (a tripeptide and a hexapeptide) were subsequently tested for effects on iron uptake with Caco-2 cell monolayers. Fractions of milk digests obtained through Sephadex G-25 gel filtration had a significant enhancing effect on iron uptake in Caco-2 cells compared to nonfractionated milk digests. Two fractions (P = 0) and the hexapeptide (P < 0.0001) enhanced iron uptake by up to 3-fold, whereas others and the tripeptide had no effect. These results suggest that selected peptides produced during the in vitro digestion of milk may enhance iron absorption; however, it remains to be demonstrated whether this effect may be nutritionally significant.     

Effect of 2alpha-hydroxyursolic acid on NF-kappaB activation induced by TNF-alpha in human breast cancer MCF-7 cells

Apples are one of the largest contributors of fruit phenolics of all fruits consumed by Americans and contain a variety of bioactive compounds, which have health benefits. Consumption of apples has been linked to reduced risk of chronic diseases such as cancer and cardiovascular disease. Apple extracts have been shown to have the capabilities of inhibiting NF-kappaB activation in human breast cancer MCF-7 cells. 2Alpha-hydroxyursolic acid is one of the major triterpenoids isolated from apple peels, and its effects on cell proliferation and TNF-alpha-induced NF-kappaB activation in MCF-7 cells were examined. 2Alpha-hydroxyursolic acid significantly inhibited MCF-7 cell proliferation at doses of 20 microM (p < 0.05). Preincubation with 2alpha-hydroxyursolic acid suppressed TNF-alpha-induced NF-kappaB activation in a dose-dependent manner and significantly inhibited the activation at a dose of 20 microM of 2alpha-hydroxyursolic acid (p < 0.05). 2Alpha-hydroxyursolic acid treatment did not affect the phosphorylation level of NF-kappaB inhibitor (IkappaB-alpha), but proteasome activity in MCF-7 cells was inhibited significantly at doses of 10 and 20 microM ( p < 0.05). These results suggest that 2alpha-hydroxyursolic acid has antiproliferative activities against MCF-7 cells and capabilities inhibiting NF-kappaB activation induced by TNF-alpha partially by suppressing proteasome activities.     

Simultaneous determination of (45)calcium and (65)zinc uptake by caco-2 cells

A simple method for simultaneously determining cell-associated Ca and Zn in Caco-2 cells is described. Calcium and zinc uptake was measured via radioisotopes (45)Ca and (65)Zn. Preliminary studies revealed that (65)Zn, a positron (beta(+)) and gamma emitter, contributed to (45)Ca counts in a liquid scintillation counter (LSC). However, (45)Ca, being a true beta emitter, did not contribute to the counts in a gamma counter (gammaC). To differentiate the counts of (45)Ca from those of (65)Zn, first a (65)Zn-labeled cell suspension was read in a gammaC and an LSC, thus obtaining the relationship between the radioactive counts obtained from the gammaC and LSC. This information defined the linear relationship between gammaC (65)Zn counts per minute (CPM) and LSC (65)Zn CPM. Because the (45)Ca and (65)Zn counts obtained in the LSC are additive, giving total LSC CPM, the value of LSC (45)Ca CPM was obtained by subtracting LSC (65)Zn CPM from total LSC CPM for the dual-labeled cell sample, obtaining then LSC (45)Ca CPM. To determine the absolute activity or disintegrations per minute (DPM) of each isotope in the dual-labeled sample, the linear relationship between DPM and CPM was determined for each isotope. The method is simple and straightforward for the determination of (45)Ca counts from a sample also containing (65)Zn, using gamma and liquid scintillation counters.     

Characterization of metabolites of hydroxycinnamates in the in vitro model of human small intestinal epithelium caco-2 cells

Hydroxycinnamic acids are antioxidant phenolic compounds which are widespread in plant foods, contribute significantly to total polyphenol intakes, and are absorbed by humans. The extent of their putative health benefit in vivo depends largely on their bioavailability. However, the mechanisms of absorption and metabolism of these phenolic compounds have not been described. In this study, we used the in vitro Caco-2 model of human small intestinal epithelium to investigate the metabolism of the major dietary hydroxycinnamates (ferulate, sinapate, p-coumarate, and caffeate) and of diferulates. The appearance of metabolites in the medium versus time was monitored, and the various conjugates and derivatives produced were identified by HPLC-DAD, LC/MS, and enzyme treatment with beta-glucuronidase or sulfatase. Enterocyte-like differentiated Caco-2 cells have extra- and intracellular esterases able to de-esterify hydroxycinnamate and diferulate esters. In addition, intracellular UDP-glucuronosyltransferases and sulfotransferases existing in Caco-2 cells are able to form the sulfate and the glucuronide conjugates of methyl ferulate, methyl sinapate, methyl caffeate, and methyl p-coumarate. However, only the sulfate conjugates of the free acids, ferulic acid, sinapic acid, and p-coumaric acid, were detected after 24 h. The O-methylated derivatives, ferulic and isoferulic acid, were the only metabolites detected following incubation of Caco-2 cells with caffeic acid. These results show that the in vitro model system differentiated Caco-2 cells have the capacity to metabolize dietary hydroxycinnamates, including various phase I (de-esterification) and phase II (glucuronidation, sulfation, and O-methylation) reactions, and suggests that the human small intestinal epithelium plays a role in the metabolism and bioavailability of these phenolic compounds.     

Transepithelial transport of chlorogenic acid, caffeic acid, and their colonic metabolites in intestinal caco-2 cell monolayers

Both chlorogenic and caffeic acids exhibited nonsaturable transport in Caco-2 cells, whereas caffeic acid also showed proton-coupled polarized absorption. Thus, the absorption efficiency of caffeic acid was greater than that of chlorogenic acid. Polarized transport of caffeic acid was inhibited by substrates of MCT such as benzoic and acetic acids. Almost all of the apically loaded chlorogenic and caffeic acid was retained on the apical side, and the transepithelial flux was inversely correlated with the paracellular permeability of Caco-2 cells. These results indicate that transport was mainly via paracellular diffusion, although caffeic acid was absorbed to a lesser extent by the monocarboxylic acid transporter (MCT). Furthermore, m-coumaric acid and 3-(m-hydroxyphenyl)propionic acid, the main metabolites of chlorogenic and caffeic acid by colonic microflora, competitively inhibited the transport of fluorescein, a known substrate of MCT. This suggests that their absorption could also be mediated by MCT. These findings have exemplified the physiological importance of MCT-mediated absorption in both phenolic acids per se and their colonic metabolites.     

Sulforaphane, erucin, and iberin up-regulate thioredoxin reductase 1 expression in human MCF-7 cells

Isothiocyanates (ITCs) found in cruciferous vegetables are potentially important anticarcinogenic phytochemicals for many types of cancers including breast cancer. In this study, we have shown that three isothiocyanates, sulforaphane, erucin, and iberin, are potent inducers of thioredoxin reductase 1 (TrxR1) in human breast cancer MCF-7 cells. Sulforaphane, erucin, and iberin at 1 microM induce TrxR1 mRNA 2-3-fold within 8 h of treatment, and induce mRNA 5-7-fold with 12 microM ITC treatments. Selenium did not affect sulforaphane-induced TrxR1 mRNA levels, but significantly enhanced both TrxR1 protein expression (up to 9-fold in erucin treatment) and corresponding activities. These results suggest that dietary ITCs are important factors in the regulation of redox status through the induction of the selenoprotein thioredoxin reductase.     

Bioavailability of calcium from milk-based formulas and fruit juices containing milk and cereals estimated by in vitro methods (solubility, dialyzability, and uptake and transport by caco-2 cells)

An adequate calcium intake during the first years of life is needed for normal growth and development and to prevent rickets. The bioavailability of calcium from infant foods (milk-based formulas and fruit juices containing milk and cereals, FMC), the dietary sources of calcium in these stages of life, has been estimated on the basis of simulated gastrointestinal digestion and calcium solubility and dialyzability values and on the efficiency of transport and uptake by Caco-2 cells. The ranking of samples according to calcium bioavailability depends on the use of solubility or dialyzability as criterion. On the basis of the former, the highest value corresponded to adapted formulas and the lowest to fruit juices. However, when using percentage dialysis, the highest value corresponded to fruit juices and the lowest to follow-up formulas. The highest percentages of transport efficiency and uptake by Caco-2 cells corresponded to calcium from the analyzed fruit juices, followed by toddler, follow-up, and adapted formulas.     

Reduction of acrylamide uptake by dietary proteins in a caco-2 gut model

The report of elevated acrylamide levels in some foods raised an international health alarm, because acrylamide probably has carcinogenic, neurotoxic, and genotoxic properties. However, data on the bioavailability of acrylamide from food matrices in humans are limited. In particular, only little is known about the interactions of acrylamide with food ingredients. Using a human intestine model (Caco-2 cells), this study shows that acrylamide monomers are highly bioavailable and pass the cell monolayer via passive diffusion. Furthermore, acrylamide binds to dietary proteins such as chicken egg albumin under intestinal and cooking conditions. This binding reduces the concentration of acrylamide monomers and leads to a reduced uptake by Caco-2 cells. Hence, it is concluded that a protein-rich diet may reduce acrylamide uptake.     

不同肥源对萝卜硝酸盐累积分布和同化的影响

采用田间试验研究了牛粪、化肥单施和配施对萝卜产量,菜体硝酸盐累积、分布、同化,及土壤硝态氮含量变化的影响。结果表明,牛粪、化肥单施和配施,萝卜产量动态变化依次为FOM(1/2化肥+1/2牛粪)、OM(牛粪)F(化肥)CK(无肥),叶部和肉质根硝酸盐含量高低依次为FFOMCKOM,粗蛋白累积量依次为FFOMCKOM,土壤硝态氮含量动态变化依次为FFOMOMCK。综合各因素总体以化肥配施牛粪最为适中,若重点考虑食用安全和土壤硝态氮累积环境效应,则以单施牛粪表现为最佳,各处理引起硝酸盐富集和土壤硝态氮残留的风险依次为化肥(1/2化肥+1/2牛粪)牛粪。萝卜硝酸盐含量分布表现为叶部生长旺盛期叶部硝酸盐含量高于肉质根,肉质根生长旺盛期地下部将贮存更多的硝酸盐。在地上部,当植株硝酸盐富集时,硝酸盐累积在内叶和叶柄中;当植株生长处于养分"饥饿"状态时,硝酸盐释放到外叶和叶片中。因此,菜地连续施用有机肥,不仅可减少蔬菜对硝酸盐的富集,且可维持后期蔬菜产量。     

长期施肥对褐潮土磷素积累、形态转化及其有效性的影响

系统研究了14年定位试验不同施肥处理对褐潮土磷素积累、形态转化及其有效性的影响,结果表明:长期不施磷肥土壤的全磷、速效磷、无机磷总量以及各组分含量较长期休闲处理均明显降低;施用磷肥的处理则相应提高。施肥对Ca2 P含量的影响最大,减少幅度最高为94 7%,几乎耗竭;施磷增加幅度最高可达34倍。其次是Ca8 P和Al P。有机肥配施磷肥更有利于土壤中积累磷素的有效性转化,转变成的Ca2 P为34 5%,明显高于单施磷肥所形成的23 1%,转变成的Ca10 P和O P(闭蓄态P)仅为7%和1 6%,明显低于单施磷肥所形成的11 4%和2 6%。     

施肥模式对茶叶产量、营养累积及土壤肥力的影响

施肥是提高茶叶产量、品质、土壤质量及促进茶园可持续利用最重要的农业措施之一。为了筛选出合理的茶树施肥模式,采用连续4周年的田间定位试验方法,研究了6种不同施肥模式(不施肥、茶树配方化肥、1/2茶树配方化肥+1/2有机肥、有机肥、茶树配方化肥+豆科绿肥、1/2茶树配方化肥+1/2有机肥+豆科绿肥)对茶叶产量,茶叶中氮、磷、钾、茶多酚和水浸出物的累积量及茶园土壤肥力状况的影响。结果表明:与对照(不施肥)模式相比,其他几种不同施肥模式均在一定程度上增加了茶叶产量,促进了茶叶营养物质的累积,并提高了茶园土壤的基本肥力状况;其中,1/2茶树配方化肥+1/2有机肥+豆科绿肥的试验效果最佳,其3年茶叶总产量最高,为5 929 kg.hm?2,比对照提高106.17%;茶叶氮、磷和钾累积量最高,分别为4.962 kg.hm?2、0.48 kg.hm?2和5.966 kg.hm?2,比对照分别提高88.6%、57.41%和98.87%;茶叶茶多酚和水浸出物累积量最高,分别为23.39 kg.hm?2和119.41 kg.hm?2,比对照分别提高73.29%和85.56%;并比对照分别提高茶园土壤有机质1.29倍、全氮1.7倍、全磷2.98倍、速效氮1.59倍、速效磷34.3倍和速效钾3.3倍。1/2茶树配方化肥+1/2有机肥+豆科绿肥施肥模式值得在茶园施肥上进一步推广应用。     

Relationships between selenium,micronutrients, carbohydrates,and alkaloid accumulation in Catharanthus roseus cells

When a culture media contained sulfur (S) or was partially depleted in S or selenium (Se) was added, the Catharanthus roseus cells had the same biomass during the stationary phase of growth. The medium partially depleted in sulfate did not change the S content of cells suggesting that they did not completely remove S from the culture medium. The addition of selenite or selenate increased zinc (Zn) and copper (Cu) accumulation but did not modify the levels of S, potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), manganese (Mn), sodium (Na), or aluminum (Al). In the partially S‐deficient medium, both the carbohydrate content and alkaloid accumulation were decreased. Selenium feeding restored or enhanced the capacity of cells to accumulate these compounds. The effects were dependent on the form and the quantity of Se added to the culture medium. Selenate was found less effective than selenite.     

氮磷钾肥对紫云英产量及养分积累的影响

通过田间试验,研究了NPK肥对紫云英生长、产量及养分积累的影响.结果表明,NPK配施能促进紫云英生长,显著提高产量和养分积累量.NPK处理(施N 75 kg/hm2 、P2O5 60 kg/hm2和K2O 60 kg/hm2)的紫云英茎数、株高和每茎复叶数分别是不施肥处理的3.77、1.81和1.60倍,是施PK处理的2.19、1.23和1.16倍,是施NK处理的2.11、1.16和1.11倍,是施NP处理的1.44、1.19和1.16倍.NPK配施的鲜草产量分别比不施肥、PK配施、NK配施及NP配施处理增加26.47、14.22、7.18和10.74 t/hm2.不同施肥处理都能使紫云英的养分积累量显著提高,其中NPK配施处理的紫云英地上部N、P2O5、K2O和C的积累量最大,分别是不施肥处理的3.66、3.27、2.85和2.80倍.试验结果说明,合理施用N、P、K肥能明显促进紫云英的生长,提高产量和养分积累量,有利于提高紫云英种植效益.     

Arsenic uptake,distribution, and accumulation in tomato plants: Effect of arsenite on plant growth and yield

The response of tomato (Lycopersicum esculentum Mill, cultivar Marmande) plants to different levels of arsenic (As) in nutrient solution was investigated—the processes of uptake, distribution and accumulation of As, and the effect of arsenite on yield and plant growth (plant height, diameter of stem, stem and root length, fresh and dry weight of root, stems, leaves, and fruit). The experiment was performed at three levels of As: 2, 5 and 10 mg/L [added as sodium arsenite (NaAsO₂)] in a nutrient solution, together with the corresponding control plants. Arsenic uptake depended on the As concentration in solution and As content in the roots increased as the time of treatment increased. The most important finding was the high toxicity of arsenite to roots. The concentration in stems, leaves, and fruit was correlated with the As level in the nutrient solution. Although the As level of 10 mg/L damaged the root membranes, resulting in a significant decrease in the upward transport of As. Arsenic exposure resulted in a drastic decrease in plant growth parameters (e.g., maximum decrease of 76.8% in leaf fresh weight) and in tomato fruit yield (maximum reduction of 79.6%). However, it is important to note that the As concentration in the fruits was not toxic or harmful for human consumption.     

施肥对蔬菜硝酸盐累积的影响研究

试验研究施肥对福州市多种蔬菜硝酸盐含量影响结果表明 ,农户传统施肥方式蔬菜硝酸盐含量为 5 9.5～374 3.1mg/kg,平均硝酸盐含量为绿叶菜类 >白菜类 >葱蒜类 >根茎类 >瓜类 >豆类 >茄果类 ,且同类不同品种蔬菜硝酸盐含量也存在差异。以WHO/FAO规定的允许值为标准评价绿叶菜类超标最重 ,其次为白菜类和葱蒜类 ,其他 4类未超标。化肥纯N施用量为 4 5 0kg/hm2 时 ,化学N肥对蔬菜硝酸盐累积的贡献率 >85 % ,其中不同品种N肥贡献率依次为NH4NO3 >NH4HCO3 >CO(NH2 ) 2 >(NH4) 2 SO4>NH4Cl。N肥施用量与蔬菜硝酸盐累积量呈正相关 ,双氰胺施用量与蔬菜硝酸盐累积量呈负相关。等N量下蔬菜硝酸盐累积量随基肥所占比例减少、追肥所占比例增大而增大 ,且随追肥后时间推移呈直线下降。配施有机氮可降低蔬菜硝酸盐含量 ,降幅施厩肥 >土杂肥。调整与优化蔬菜品种及施肥结构 ,采用“重头、稳中、控尾”施N方式 ,根据蔬菜食用卫生要求选择N肥用量及其使用安全期与双氰胺用量 ,可降低蔬菜硝酸盐含量。