Vaccenic acid (<Emphasis Type="Italic">t</Emphasis>11–18∶1) is converted to <Emphasis Type="Italic">c</Emphasis>9,<Emphasis Type="Italic">t</Emphasis>11-CLA in MCF-7 and SW480 cancer cells

The aims of this study were to determine whether vaccenic acid (VA; t11–18∶1) is converted to c9,t11-CLA in human mammary (MCF-7) and colon (SW480) cancer cell lines and whether VA influences cell viability and other CLA-bioresponsive markers. When cells were incubated in the presence of VA at concentrations of 5 to 20 μg/mL, both VA and c9,t11-CLA increased in cellular lipids in a dose-dependent manner. After 4 d of incubation of SW480 and MCF-7 cells with VA (20 μg/mL), c9,t11-CLA increased from undetectable levels to 8.57 and 12.14 g/100 g FAME in cellular lipids, respectively. VA supplementation for 4 d at 5, 10, and 15 μg/mL had no effect on cell growth, whereas 20 μg/mL significantly (P<0.05) reduced cell growth in both cell lines. VA (20 μg/mL) treatment induced DNA fragmentation and significantly (P<0.05) depeleted cytosolic GSH levels in the SW480 cell line after 4 d of incubation, suggesting that apoptosis was the mode of cell death induced by VA. Both VA and c9,t11-CLA reduced (P<0.05) total ras expression in SW480 cells. ¹⁴C-Arachidonic acid uptake into the MG fraction was significantly increased (P<0.05) in both cell lines while uptake into the phospholipid fraction decreased in response to VA. VA treatment significantly (P<0.05) increased 8-epi-prostaglandin F_2α in both cell lines. The data indicate that growth suppression and cellular responses of both cells lines are likely mediated by VA desaturation to c9,t11-CLA via Δ⁹-desaturase.     

Comparison of linoleic acid and eicosapentaenoic acid incorporation into human breast cancer cells

To gain some insight into the mechanisms involved in the opposing effects of linoleic acid (LA) and eicosapentaenoic acid (EPA) on the growth and invasiveness of MDA-MB-435 human breast cancer cells, the dynamics of the uptake by cells and the incorporation of [¹⁴C]LA and [¹⁴C]EPA into major lipid and phospholipid pools, as well as the effects of unlabeled EPA or LA on the uptake and distribution of [¹⁴C]LA or [¹⁴C]EPA, respectively, were examined. Cells were exposed to [¹⁴C]LA (1.28 μg/mL) or [¹⁴C]EPA (1.0 μg/mL) and unlabeled EPA or LA, respectively, at 0, 1, 4 and 16 μg/mL for 24 h in serum-free media. The uptake of each fatty acid (FA) was linear over time and was not affected by the presence of the opposing FA. For both FA, 80–90% was incorporated into the phospholipid fraction with the remaining 10–20% in neutral lipids. The relative distribution profile of [¹⁴C]LA among the phospholipid classes indicated a preferential incorporation into phosphatidylcholine (65%), whereas [¹⁴C]EPA was mostly found in phosphatidylethanolamine (58%). In the presence of unlabeled EPA or LA at various concentrations, corresponding dose-dependent shifts of [¹⁴C]LA or [¹⁴C]EPA from the phospholipid to the neutral lipid pool were noted, which did not alter the relative distribution of the FA among the phospholipid classes. Exogenous exposure to EPA or LA increased its content in membrane phospholipids while concurrently decreasing LA or EPA content, respectively, in a dose-dependent manner. Arachidonic acid content of membrane phospholipids remained constant. The divergent distribution profiles of LA and EPA within the phospholipid compartment provides some insight into the mechanisms of their opposing effects on MDA-MB-435 cell growth and invasiveness. Also, the effects of LA and EPA on the uptake and distribution of their opposing FA shed some light on the mechanisms mediating their competitive effects.     

EPA,but not DHA,decreases mean platelet volume in normal subjects

The first indication of platelet activation is an increase in mean platelet volume (MPV). n−3 FA are known to inhibit platelet function and to reduce the risk for coronary heart disease. The purpose of this study was to determine the effects of FPA and DHA on MPV. Healthy subjects received olive oil placebo for 4 wk and then were randomly assigned to receive 4g of ethyl esters of either safflower oil (n=11), EPA (n=10), or DHA (n=12) for 4 wk. At the end of placebo run-in and treatment periods, MPV (fL; mean±SEM) and platelet count (PLT-CT; 10³/μL blood) were measured in the basal state and after ex vivo stimulation with collagen (10 μg/mL), cold (4°C), and heat (37°C). Unlike DHA, EPA lowered MPV as compared with safflower oil (7.2±0.1 vs. 7.5±0.1 fL; P<0.05) and raised PLT-CT (211±18 vs. 192±18 10³/μL; P<0.05) in the fasting state. Collagen and cold significantly increased MPV whereas heat lowered MPV regardless of treatment. All stimuli decreased PLT-CT. EPA significantly increased platelet EPA (0.2±0.1 vs. 3.3±0.4%) and docosapentaenoic acid (DPA; 2.2±0.3 vs. 2.9±0.3%) concentrations, but not DHA. DHA treatment significantly increased DHA (1.4±0.2 vs. 4.1±0.5%) and DPA (2.0±0.4 vs. 3.0±0.4%) concentrations, but not EPA. In conclusion, EPA, but not DHA, reduces platelet activation, an early step in platelet aggregation.     

Polyunsaturated fatty acids increase the sensitivity of 36B10 rat astrocytoma cells to radiation-induced cell kill

Polyunsaturated fatty acids (PUFA) such as γ-linolenic acid (GLA, 18:3n-6), eicosapentaenoic acid (EPA, 20: 5n-3), and docosahexaenoic acid (DHA, 22:6n-3) have been shown to be cytotoxic to tumor cells. The objective of this work was to study the effect of PUFA on the radiation response of a 36B10 rat astrocytoma cell line. Supplementation of the astrocytoma cells with 15–45 μM GLA, EPA, or DHA produced marked changes in the fatty acid profiles of their phospholipids and neutral lipids. The methylene bridge index of these lipids increased significantly. These PUFA also exerted cytotoxic effects, as determined using the clonogenic cell survival assay. While GLA and DHA produced a moderate cell-killing effect, EPA was extremely cytotoxic, especially at a concentration of 45 μM. The monounsaturated oleic acid (OA, 18:1n-9) did not affect cell survival. Further, all three PUFA, and particularly GLA, increased the radiation-induced cell kill; OA did not enhance the effect of radiation. α-Tocopherol acetate blocked the enhanced radiation sensitivity of GLA- and DHA-supplemented cells. In conclusion, GLA, EPA, and DHA supplementation prior to, during, and after irradiation can enhance the radiation-induced cytotoxicity of rat astrocytoma cells. GLA and DHA supplementation post-irradiation also enhanced the radiation response of the 36B10 cells. Because GLA maximally increases the radioresponsiveness of a rat astrocytoma, this PUFA might prove useful in increasing the therapeutic efficacy of radiation in the treatment of certain gliomas.     

Eicosapentaenoic acid inhibits tube formation of vascular endothelial cellsin vitro

We previously have described a quantitative angiogenesisin vitro model, in which endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures. Using this model, the effect of eicosapentaenoic acid (20∶5n−3) on tube formation was investigated. When the endothelial cells isolated from bovine carotid artery were treated for 2 days with 5 μg/mL of arachidonic acid (20∶4n−6), eicosapentaenoic acid or docosahexaenoic acid (22∶6n−3), these polyunsaturated fatty acids were extensively incorporated into cellular phospholipids. The content of arachidonic, eicosapentaenoic and docosahexaenoic acid increased from 9.58% to 23.29%, from 0.98% to 11.76% and from 6.88% to 18.40%, respectively. When the eicosapentaenoic acid-treated cells were cultured between collagen gels, the tube-forming ability of the cells was markedly inhibited. The inhibition was dose-dependent between 1.0 and 5.0 μg/mL of eicosapentaenoic acid. At 5.0 μg/mL of eicosapentaenoic acid the inhibition reached 76%. By contrast, arachidonic acid increased tube formation, and docosahexaenoic acid had no effect. To elucidate the mechanism of eicosapentaenoic acid induced inhibition ofin vitro tube formation, we examined the effect of the acid on the proliferation of endothelial cells. Eicosapentaenoic acid at any dose (<5.0 μg/mL) had no effect on the proliferation of endothelial cells cultured on plastic plates without collagen gel. However, when the cells were cultured between collagen gels, eicosapentaenoic acid inhibited cell growth in a dose-dependent manner with maximum inhibition being observed at 2.5 μg/mL. These data suggest that eicosapentaenoic acid suppresses tube formation of endothelial cells, at least in part,via its inhibitory effect on cellular proliferation. Thus eicosapentaenoic acid may act as an endogenous inhibitor of angiogenesis under various pathological conditions, including tumor growth and chronic inflammation.     

Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status

Potential antiproliferative effects of tocotrienols, the major vitamin E component in palm oil, were investigated on the growth of both estrogen-responsive (ER+) MCF7 human breast cancer cells and estrogen-unresponsive (ER-) MDA-MD-231 human breast cancer cells, and effects were compared with those of α-tocopherol (αT). The tocotrienol-rich fraction (TRF) of palm oil inhibited growth of MCF7 cells in both the presence and absence of estradiol with a nonlinear dose-response but such that complete suppression of growth was achieved at 8 μg/mL. MDA-MB-231 cells were also inhibited by TRF but with a linear dose-response such that 20 μg/mL TRF was needed for complete growth suppression. Separation of the TRF into individual tocotrienols revealed that all fractions could inhibit growth of both ER+ and ER- cells and of ER+ cells in both the presence and absence of estradiol. However, the γ- and δ-fractions were the most inhibitory. Complete inhibition of MCF7 cell growth was achieved at 6 μg/mL of γ-tocotrienol/δ-tocotrienol (γT₃/δT₃) in the absence of estradiol and 10μm/mL of δT₃ in the presence of estradiol, whereas complete suppression of MDA-MB-231 cell growth was not achieved even at concentrations of 10μg/mL of δT₃. By contrast to these inhibitory effects of tocotrienols, αT had no inhibitory effect on MCF7 cell growth in either the presence or the absence of estradiol, nor on MDA-MB-231 cell growth. These results confirm studies using other sublines of human breast cancer cells and demonstrate that tocotrienols can exert direct inhibitory effects on the growth of breast cancer cells. In searching for the mechanism of inhibition, studies of the effects of TRF on estrogen-regulated pS2 gene expression in MCF7 cells showed that tocotrienols do not act via an estrogen receptor-mediated pathway and must therefore act differently from estrogen antagonists. Furthermore, tocotrienols did not increase levels of growth-inhibitory insulin-like growth factor binding proteins (IGFBP) in MCF7 cells, implying also a different mechanism from that proposed for retinoic acid inhibition of estrogen-responsive breast cancer cell growth. Inhibition of the growth of breast cancer cells by tocotrienols could have important clinical implications not only because tocotrienols are able to inhibit the growth of both ER+ and ER- phenotypes but also because ER+ cells could be growth-inhibited in the presence as well as in the absence of estradiol. Future clinical applications of TRF could come from potential growth suppression of ER+ breast cancer cells otherwise resistant to growth inhibition by antiestrogens and retinoic acid.     

Production of eicosapentaenoic acid from marine bacteria

A marine bacterium, judged as a new species close toShewanella putrefaciens, was isolated from the intestinal contents of the Pacific mackerel. The isolated strain SCRC-2378 produced eicosapentaenoic acid (EPA) as the sole polyunsaturated fatty acid, which amounted to 24–40% of the total fatty acid in the cell, which corresponded to 2% of dry cell weight. Under the optimal growth conditions (pH 7.0, 20°C, and grown aerobically for 12–18 h), the yield of SCRC-2738 reached 15 g of dry cells/L or 2×10¹⁰ viable cells/mL. EPA existed as phospholipid and was found in thesn-2 position of phosphatidylethanolamine and phosphatidylglycerol. The 38 kbp (1,000 base pairs) genome DNA fragment was cloned from SCRC-2738 and expressed inEscherichia coli, which resulted in the production of EPA. The nucleotide sequence of the 38 kbp DNA fragment was determined. The DNA fragment contains eight open reading frames, and three of them posses homology with enzymes involved in fatty acid synthesis. Thus, it may be possible that these EPA biosynthesis genes are applied for EPA production in yeasts or higher plants, and offer a new method for EPA synthesis as new foods containing EPA.     

Incorporation of oleic acid and eicosapentaenoic acid into glycerolipids of cultured normal human fibroblasts

Confluent skin fibroblasts from normal humans were incubated in serum free medium with up to 100 nmole/mL eicosapentaenoic acid (EPA; bound to albumin in a 4.6∶1 ratio) and compared with cells incubated with oleic acid (OA) at similar concentrations. The rate of [¹⁴C]OA incorporation into triacylglycerol (TG) (nmol/mg/h) was approximately 5-fold greater than that of [¹⁴C]EPA. The mass of TG formed after incubation of fibroblasts with EPA was also significantly lower than that formed with OA (43.2 ±9.3vs. 59.5±6.6 μg/mg cell protein, respectively,P=0.006). The addition of excess, unlabeled EPA reduced the rate of incorporation of [¹⁴C]OA into TG whereas unlabeled OA stimulated incorporation of [¹⁴C]EPA into TG. When the cells were preincubated with human serum basic proteins (BP I, II and III), the mass of TG formed (compared to baseline) was significantly higher with the basic proteins whether OA or EPA was studied. Only BP I significantly stimulated the mass of cell phospholipids, an effect that occurred with either OA or EPA in the medium. The results suggest that in cultured normal human fibroblasts, OA is a better substrate for TG synthesis than EPA and that this effect may be accentuated by the presence of the basis proteins.     

Inhibition of 12- and 15-lipoxygenase activities and protection of human and tilapia low density lipoprotein oxidation by I-Tiao-Gung (<Emphasis Type="Italic">Glycine tomentella</Emphasis>)

I-Tiao-Gung, Glycine tomentella, has been used extensively as a traditional herbal medicine to relieve physical pain, but its bioactivity has not been studied systematically. Ninety-five percent ethanol extracts of G. tomentella (GT-E) showed antioxidant activity in human plasma by prolonging the lag phase (+T_lag) of Cu²⁺-induced, LDL oxidation and were dose dependent. The+T_lag of LDL combined with 3.2 μg/mL GT-E was similar to that with 2.0 μM (ca. 0.5 μg/mL) Trolox. A similar inhibitory effect was found toward tilapia plasma LDL. In addition, GT-E inhibited tilapia thrombocyte (nucleated platelet), 5-, 12-, and 15-lipoxygenase (LOX). The IC₅₀ values were 0.43, 0.72, and 0.42 μg/mL, respectively, whereas the IC₅₀ values for nordihydroguaiaretic acid (NDGA) on 5-, 12-, and 15-LOX were 2.3, 1.6, and 1.7 μg/mL, respectively. The IC₅₀ value for cyclooxygenase-2 (COX-2) inhibition by GT-E was 42.0 μg/mL, whereas the IC₅₀ value by indomethacin as a positive control was 0.61 μg/mL. The prevention of LDL oxidation and the dual inhibition of LOX and COX-2 are indicative of the possible roles of I-Tiao-Gung in antiatherosclerosis and anti-inflammation.     

Eicosapentaenoic acid promotes apoptosis in Ramos cells <Emphasis Type="Italic">via</Emphasis> activation of caspase-3 and -9

Eicosapentaenoic acid (EPA; 20∶5n−3) may reduce the cell number in cultured leukemia/lymphoma cells owing to reduced cell proliferation, induction of cell death, or a combination of these processes. EPA has been shown to promote apoptosis in Ramos cells, and our present study was focused on a possible cell cycle arrest and the pathways by which the apoptotic process is induced. Apoptosis may proceed along the intrinsic (mitochondrial) or the extrinsic (death receptor) pathway, which are mediated via different caspases. Caspases are a class of homologous cysteine proteases recognized as pivotal mediators of apoptosis. We investigated whether EPA affects progression of the cell cycle or promotes apoptosis directly. By incorporation of [³H]thymidine and [³H]valine, we showed that DNA, as well as protein synthesis, was reduced after incubation of Ramos cells with EPA for 6h. We monitored cell cycle distribution by 5-bromo-2′-deoxyuridine staining and observed no cell cycle arrest in the EPA-incubated cells. Incubation of cells with EPA caused PS-flipping, as demonstrated by annexin V-binding (flow cytometry), and cleavage of poly(ADP-ribose) polymerase measured by Western blot analysis. Furthermore, we observed increased activity of caspase-3 and-9, but not of caspase-8. Whereas inhibitors of caspase-3 and-9 reduced EPA-induced apoptosis, inhibition of caspase-8 did not. This suggests that EPA may promote apoptosis via the intrinsic pathway in Ramos cells. Thus, the reduction in cell number can be explained by a direct apoptotic effect of EPA rather than via cell cycle arrest.     

Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3-L1 preadipocytes

Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt. 1), cells were cultured in media containing a crude mixture of CLA isomers or pure LA at 0, 10, 50, or 200 μM for 4 d. Preadipocyte proliferation (cell number, ³H-thymidine incorporation into DNA) decreased as the level of CLA increased in the cultures. In contrast, LA had no impact on DNA synthesis. In Experiment 2a, postconfluent cultures were grown in media containing a crude mixture of CLA isomers or LA at 0, 10, 50, or 200 μM for the next 6 d. Postconfluent cultures supplemented with 50–200 μM CLA had less triglyceride (TG) and were smaller in size than cultures supplemented with similar amounts of LA. In Experiment 2b, postconfluent cultures supplemented with 200 μM of a crude mixture of CLA isomers or LA were harvested on days 1, 3, 6, or 9. Differences in TG content of cultures supplemented with 200 μM CLA compared to control and LA-supplemented cultures became apparent after 3 d of culture. Experiments 3a and 3b examined whether the fatty acid vehicle (BSA vs. ethanol) or the vitamin E status (±0.2 mM α-tocopherol) of the cultures altered CLA’s impact on preadipocyte TG content. In Experiment 3a, ethanol-treated cultures had more TG than non-ethanol-treated cultures regardless of the fatty acid treatment. In Experiment 3b, cultures treated with 100 μM of either a crude mixture of CLA or the trans-10, cis-12 CLA isomer without supplemental vitamin E for 6 d had less TG than CLA-treated cultures containing vitamin E. In Experiment 4, postconfluent cultures were grown in media containing 100 μM LA or either a crude mixture of CLA isomers or the trans-10, cis-12 CLA isomer for 24–96 h to assess CLA’s influence on the cell cycle and indices of apoptosis. Cultures treated with 100 μM CLA for 24–96 h had more apoptotic cells than BSA- or LA-treated cultures. Furthermore, cultures treated for 48 h with CLA had fewer cells in the S-phase than control cultures. The effects of the trans-10,cis-12 CLA isomer were more pronounced than those of the crude mixture of CLA isomers. These data suggest that CLA may exert its antiobesity effects by inhibiting proliferation, attenuating TG content, and/or inducing apoptosis in (pre)adipocytes.     

Growth and eicosapentaenoic acid production byPhaeodactylum tricornutum in batch and continuous culture systems

Maximum specific growth rate (μ_max) ofPhaeodactylum tricornutum increased with increasing culture reactor surface-to-volume ratio. Values for μ_max of 0.647, 0.377 and 0.339 day⁻¹ were observed for the 75-mL tube, 5.6-L tank and the 16-L tank, respectively. Higher biomass was achieved in the 75-mL batch culture tube under continuous light as compared with light cycle conditions. Palmitic acid, palmitoleic acid and eicosapentaenoic acid (EPA) accounted for over 60% of total fatty acids in the batch tube culture, with EPA content increasing to a maximum after three days. In chemostat cultures, run at dilution rates of 0.15 day⁻¹ (0.45 of μ_max) and 0.3 day⁻¹ (0.9 of μ_max), cell concentration reached a steady state of 2.18 and 0.7 g/L, respectively, while contents of EPA per liter of culture at steady state were 100.9 and 82.5 mg/L, respectively. At both dilution rates, EPA content of total fatty acids was the same (35.0–35.2%). At a dilution rate of 0.3 day⁻¹, the continuous culture system manifested productivities of 0.51 g/L/d and 25.1 mg/L/d for biomass and EPA, respectively.     

Increasing DHA and EPA Concentrations Prolong Action Potential Durations and Reduce Transient Outward Potassium Currents in Rat Ventricular Myocytes

The goal of this study was to determine the mechanisms of n-3 polyunsaturated fatty acids (n-3 PUFA) on anti-arrhythmias and prevention of sudden death. The calcium-tolerant Sprague–Dawley rat ventricular myocytes were isolated by enzyme digestion. Effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on action potentials and transient outward potassium currents (I _to) of epicardial ventricular myocytes were investigated using whole-cell patch clamp techniques. Action potential durations (APDs) and I _to were observed in different concentrations of DHA and EPA. APD₂₅, APD₅₀, and APD₉₀ with 0.1 μmol/L DHA and EPA were prolonged less than 15% and 10%. However, APDs were prolonged in concentration-dependent manners when DHA and EPA were more than 1 μmol/L. APD₂₅, APD₅₀, and APD₉₀ were 7.7 ± 2.0, 21.2 ± 3.5, and 100.1 ± 9.8 ms respectively with 10 μmol/L DHA, and 7.2 ± 2.5, 12.8 ± 4.2, and 70.5 ± 10.7 ms respectively with 10 μmol/L EPA. I _to currents were gradually reduced with the increased concentrations of DHA and EPA from 1 to 100 μmol/L, and their half-inhibited concentrations were 2.3 ± 0.2 and 3.8 ± 0.6 μmol/L. The results showed APDs were prolonged and I _to current densities were gradually reduced with the increased concentrations of DHA and EPA. The anti-arrhythmia mechanisms of n-3 PUFA are complex, however, the effects of n-3 PUFA on action potentials and I _to may be one of the important mechanisms.     

Differential effects of eicosapentaenoic acid and docosahexaenoic acid on human skin fibroblasts

To better understand the mode of action of ω3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 μM oleic acid linoleic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the effects on membrane composition, fluorescence polarization and enzyme activities were followed. The cells were enriched with EPA and DHA up to 7 and 13% of total lipids, respectively, of which >95% was associated with phospholipids. In addition, the concentration of 22∶5n−3 increased with both EPA and DHA to 7.5, and 2.1% of the total fatty acids, respectively. When compared to controls (oleic acid), cells treated with DHA showed a decrease in cholesterol, phospholipids, arachidonic acid (AA) and free cholesterol/phospholipid ratio (P<0.05). In the presence of EPA, only decreases in AA and cholesterol were significant (P<0.05). Membrane fluidity, assessed by fluorescence anisotropy, was increased 16% in cells enriched with DHA (P<0.05), but showed no change with EPA or linoleic acid. There was an increase in membrane-associated 5′-nucleotidase (+27%) and adenylate cyclase (+19%) activities (P<0.05), in DHA-enriched, but not in EPA-enriched cells, when compared with oleate controls. The studies show that incorporation of DHA, but not EPA, into cell membranes of fibroblasts alters membrane biophysical characteristics and function. We suggest that these two major n−3 fatty acids of fish oils have differential effects on cell membranes, and this may be related to the known differences in their physiological effects.     

A Preliminary Assessment of Silver Nanoparticle Inhibition of Monkeypox Virus Plaque Formation

The use of nanotechnology and nanomaterials in medical research is growing. Silver-containing nanoparticles have previously demonstrated antimicrobial efficacy against bacteria and viral particles. This preliminary study utilized an in vitro approach to evaluate the ability of silver-based nanoparticles to inhibit infectivity of the biological select agent, monkeypox virus (MPV). Nanoparticles (10–80 nm, with or without polysaccharide coating), or silver nitrate (AgNO₃) at concentrations of 100, 50, 25, and 12.5 μg/mL were evaluated for efficacy using a plaque reduction assay. Both Ag-PS-25 (polysaccharide-coated, 25 nm) and Ag-NP-55 (non-coated, 55 nm) exhibited a significant (P ≤ 0.05) dose-dependent effect of test compound concentration on the mean number of plaque-forming units (PFU). All concentrations of silver nitrate (except 100 μg/mL) and Ag-PS-10 promoted significant (P ≤ 0.05) decreases in the number of observed PFU compared to untreated controls. Some nanoparticle treatments led to increased MPV PFU ranging from 1.04- to 1.8-fold above controls. No cytotoxicity (Vero cell monolayer sloughing) was caused by any test compound, except 100 μg/mL AgNO₃. These results demonstrate that silver-based nanoparticles of approximately 10 nm inhibit MPV infection in vitro, supporting their potential use as an anti-viral therapeutic.     

Investigations on the cellular uptake of hexadecylphosphocholine

The uptake of [(9,10)-³H]hexadecylphosphocholine (HePC) in six tumor cell lines was studied. All cell lines incorporated HePC in similar amounts, with the exception of the epidermoid cancer cell line KB, which took up higher amounts of HePC. The uptake of HePC at 37°C was shown to be time and concentration dependent. At 20°C, uptake was drastically reduced and at 4°C it was blocked completely. Binding of HePC, at 4°C, was not saturable at concentrations between 5 βg/mL (11.8 μM) and 100 μg/mL (235.3 μM), indicating that cell surface binding is not receptor-mediated. Furthermore, the effects of inhibitors of endocytosis were investigated. We observed a pronounced inhibitory effect by monensin and cytochalasin B. Colchicine was somewhat less effective whereas chloroquine was almost without effect. From these data we conclude that uptake of HePC is most probably mediatedvia a receptor-independent endocytotic mechanism.     

Effect of tocotrienols on the growth of a human breast cancer cell line in culture

The tocotrienol-rich fraction (TRF) of palm oil consists of tocotrienols and some α-tocopherol (α-T). Tocotrienols are a form of vitamin E having an unsaturated side-chain, rather than the saturated side-chain of the more common tocopherols. Because palm oil has been shown not to promote chemically-induced mammary carcinogenesis, we tested effects of TRF and α-T on the proliferation, growth, and plating efficiency (PE) of MDA-MB-435 estrogen-receptor-negative human breast cancer cells. TRF inhibited the proliferation of these cells with a concentration required to inhibit cell proliferation by 50% of 180 μg/mL, whereas α-T had no effect at concentrations up to 1000 μg/mL as measured by incorporation of [³H]thymidine. The effects of TRF and α-T also were tested in longer-term growth experiments, using concentrations of 180 and 500 μg/mL. We found that TRF inhibited the growth of these cells by 50%, whereas α-T did not. Their effect on the ability of these cells to form colonies also was studied, and it was found that TRF inhibited PE, whereas α-T had no effect. These results suggest that the inhibition is due to the presence of tocotrienols in TRF rather than α-T. Based on a paper presented at the PORIM International Palm Oil Congress (PIPOC) held in Kuala Lumpur, Malaysia, September 1993.     

Cell proliferation’ differentiation’ and apoptosis are modified by n−3 polyunsaturated fatty acids in normal colonic mucosa

Supplementation with low doses of eicosapentaenoic (EPA) or docosahexaenoic (DHA) acid was used here to investigate changes in epithelial proliferation’ differentiation’ and apoptosis in normal rat colonic mucosa. ACI/T rats received by oral administration low doses of purified EPA or DHA ethyl esters (1g/kg body weight) and colonic mucosa was analyzed for cell proliferation’ differentiation’ and apoptosis. n−3 Polyunsaturated fatty acid incorporation into membrane phospholipids was investigated as reflections of fatty acid metabolism. Both EPA and DHA suppressed colonocyle proliferation and increased the numbers of differentiating and apoptotic cells without modification of the crypt morphology and the number of cells per crypt columns. A significant incorporation of the supplemented fatty acids into total phospholipids was observed. This enrichment was accompanied by a decreased content in arachidonic acid. The observation that EPA and DHA do not alter crypt morphology although they modify cell turnover in normal colonic mucosa suggests a possible use of these fatty acids as dietary chemopreventive agents.     

Biocompatibility of Graphene Oxide

Herein, we report the effects of graphene oxides on human fibroblast cells and mice with the aim of investigating graphene oxides' biocompatibility. The graphene oxides were prepared by the modified Hummers method and characterized by high-resolution transmission electron microscope and atomic force microscopy. The human fibroblast cells were cultured with different doses of graphene oxides for day 1 to day 5. Thirty mice divided into three test groups (low, middle, high dose) and one control group were injected with 0.1, 0.25, and 0.4 mg graphene oxides, respectively, and were raised for 1 day, 7 days, and 30 days, respectively. Results showed that the water-soluble graphene oxides were successfully prepared; graphene oxides with dose less than 20 μg/mL did not exhibit toxicity to human fibroblast cells, and the dose of more than 50 μg/mL exhibits obvious cytotoxicity such as decreasing cell adhesion, inducing cell apoptosis, entering into lysosomes, mitochondrion, endoplasm, and cell nucleus. Graphene oxides under low dose (0.1 mg) and middle dose (0.25 mg) did not exhibit obvious toxicity to mice and under high dose (0.4 mg) exhibited chronic toxicity, such as 4/9 mice death and lung granuloma formation, mainly located in lung, liver, spleen, and kidney, almost could not be cleaned by kidney. In conclusion, graphene oxides exhibit dose-dependent toxicity to cells and animals, such as inducing cell apoptosis and lung granuloma formation, and cannot be cleaned by kidney. When graphene oxides are explored for in vivo applications in animal or human body, its biocompatibility must be considered.     

Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.