Docosahexaenoic Acid Exposure Research Articles

IPSC-Derived LCHADD Retinal Pigment Epithelial Cells Are Susceptible to Lipid Peroxidation and Rescued by Transfection of a Wildtype AAV-HADHA Vector.

Progressive choroid and retinal pigment epithelial (RPE) degeneration causing vision loss is a unique characteristic of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), a fatty acid oxidation disorder caused by a common c.1528G>C pathogenic variant in HADHA, the α subunit of the mitochondrial trifunctional protein (TFP). We established and characterized an induced pluripotent stem cell (iPSC)-derived RPE cell model from cultured skin fibroblasts of patients with LCHADD and tested whether addition of wildtype (WT) HAHDA could rescue the phenotypes identified in LCHADD-RPE. We constructed an rAAV expression vector containing 3' 3xFLAG-tagged human HADHA cDNA under the transcriptional control of the cytomegalovirus (CMV) enhancer-chicken beta actin (CAG) promoter (CAG-HADHA-3XFLAG). LCHADD-RPE were cultured, matured, and transduced with either AAV-GFP (control) or AAV-HADHA-3XFLAG. LCHADD-RPE express TFP subunits and accumulate 3-hydroxy-acylcarnitines, cannot oxidize palmitate, and release fewer ketones than WT-RPE. When LCHADD-RPE are exposed to docosahexaenoic acid (DHA), they have increased oxidative stress, lipid peroxidation, decreased viability, and are rescued by antioxidant agents potentially explaining the pathologic mechanism of RPE loss in LCHADD. Transduced LCHADD-RPE expressing a WT copy of TFPα incorporated TFPα-FLAG into the TFP complex in the mitochondria and accumulated significantly less 3-hydroxy-acylcarnitines, released more ketones in response to palmitate, and were more resistant to oxidative stress following DHA exposure than control. iPSC-derived LCHADD-RPE are susceptible to lipid peroxidation mediated cell death and are rescued by exogenous HADHA delivered with rAAV. These results are promising for AAV-HADHA gene addition therapy as a possible treatment for chorioretinopathy in patients with LCHADD.

Interaction of n-3 polyunsaturated fatty acids with host CD36 genetic variant for gut microbiome and blood lipids in human cohorts

Previous studies suggest an interaction of CD36 genetic variant rs1527483 with n-3 polyunsaturated fatty acids (PUFAs) to modulate blood lipids. However, successful replication is lacking and the role of gut microbiome remains unclear. Here, we aimed to replicate these gene-diet interactions on blood lipids and investigate their possible associations with gut microbiome. We evaluated the n-3 PUFA-rs1527483 interaction on blood lipids in two population-based cohorts (n=4,786). We profiled fecal microbiome and short-chain fatty acids among 1,368 participants. The associations between n-3 PUFAs and bacterial alpha-diversity, taxonomies and short-chain fatty acids by rs1527483 genotypes were analyzed using regression models. CD36 rs1527483-GG carriers responded better to high n-3 PUFA exposure; higher blood HDL-C (beta (95% CI): 0.05 (0.01, 0.08) mmol/L) and lower TG (log-transformed, beta (95% CI):-0.08 (-0.14,-0.02)) were observed among participants whose n-3 PUFA exposure ranked in the top quartile comparing with those in the bottom quartile. We identified docosahexaenoic acid (DHA) as the driven individual n-3 PUFA biomarker, which showed interaction with rs1527483. Among the rs1527483-GG carriers, but not other genotype groups, DHA exposure was positively associated with bacterial Faith's phylogenetic diversity, Observed OTUs, Shannon's diversity index, Dorea, Coriobacteriales Incertae Sedis spp, and fecal propionic acid levels. Another independent longitudinal cohort validated the DHA-rs1527483 interaction on gut microbiome. The identified microbial features were correlated with blood lipids, and the host biosynthesis and metabolism pathways of bile acids and aromatic amino acids. The present study found that higher n-3 PUFAs were associated with improved blood lipids and gut microbial features only among rs1527483-GG carriers. These findings highlight a potential role of gut microbiome to link the CD36 genetic variant, n-3 PUFAs and blood lipids, revealing a new research direction to interpret the gene-diet interaction for cardiometabolic health.

Fatty acid compositions of immature and mature testis are differently responsive to dietary docosahexasenoic acid during development in rats exposed to prenatal ethanol

BackgroundEthanol (EtOH) exposure impairs, but docosahexaenoic acid (DHA) supports testis functions. This study investigated whether dietary DHA and prenatal EtOH exposure affected fatty acid profiles equally in immature and mature testis during developmental stages. MethodsFemale rats were exposed to ± EtOH (3g/kg BW, twice a day via gavage) throughout pregnancy, while consuming a diet supplemented ± DHA (1.4%, w/w). Pups were continued on their mother's diet after weaning with testes collected for fatty acid analysis at different stages of reproductive development, at gestational day 20 (GD20) and postnatal day (PD) 4, 21, 49, and 90, to present fetal, neonatal, weaning, prepubertal and adult stages, respectively. ResultsRegardless of EtOH exposure, dietary DHA significantly increased in testis DHA at all ages, with testis at weaning and prepuberty being more responsive to the diet (p<0.0002). Immature testis at GD20 and PD4 contained more DHA than n-6 docosapentaenoic acid (n-6 DPA) compared to mature testis while being well responsive to the maternal DHA diet through gestation and lactation. The level of n-6 very long chain fatty acids and (VLCFA) and n-6 DPA, distinctively increased from weaning and prepuberty, respectively, and were not reduced by the DHA diet at prepuberty and adulthood. Prenatal EtOH minimally affected testis fatty acids during development. ConclusionImmature and mature testis responds differently to dietary DHA. The age around sexual maturity might be a critical time for dietary intervention as testis was more responsive to diet at this time point. The increase in DPA and n-6 VLCFA in matured testis while not affected by dietary DHA, indicates their critical roles in male reproductive function in rodents.

Effect of Prenatal Docosahexaenoic Acid Supplementation on Blood Pressure in Children With Overweight Condition or Obesity

The blood pressure-lowering property of docosahexaenoic acid (DHA) in children and adults is known, and an observational study from the Netherlands has linked higher intrauterine DHA exposure to lower childhood blood pressure. However, the association of prenatal intake of DHA supplement with childhood blood pressure has not been evaluated in randomized clinical trials. To determine the effect of DHA supplementation during pregnancy on childhood blood pressure. This prespecified secondary analysis of the Kansas University DHA Outcome Study (KUDOS), a phase 3, double-blind, randomized, placebo-controlled clinical trial was conducted at several local hospitals in the Kansas City, Kansas, metropolitan area. Pregnant women (n = 350) were enrolled in the KUDOS trial between January 10, 2006, and November 17, 2009, and were followed up until their children were 18 months of age. During pregnancy, the women received either 3 capsules per day of placebo or 600 mg per day of DHA from a mean (SD) of 14.5 (3.7) weeks' (all before 20 weeks) gestation until birth. The parents of 190 children consented to additional follow-up of their children until 6 years, which ended April 29, 2016. Study personnel involved in testing were blind to the randomization until all children had completed the trial. Data analysis was performed from May 23, 2017, to July 10, 2018. Pregnant women were assigned to either 600 mg per day of DHA or a placebo that was half soy and half corn oil. Both placebo and DHA were provided in 3 capsules per day. Childhood blood pressure was a planned secondary outcome of a study powered to measure cognitive development. The hypothesis was that DHA would lower blood pressure prior to data analysis. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured at 4, 4.5, 5, 5.5, and 6 years and were analyzed for possible covariates using mixed models to generate a final model. In total, 171 children (88 [51.5%] female) were included in this analysis. Of these children, 89 (52.0%) were randomized to the DHA group and 82 (47.9%) to the placebo group. A statistically significant interaction was found between treatment (placebo or DHA) and child weight status (5-year body mass index ≤85th or >85th percentile) for both SBP and DBP. Children who were overweight or obese whose mothers received placebo during pregnancy had higher SBP and DBP compared with children who were overweight or obese whose mothers received DHA (mean [SE] SBP, 104.28 [1.37] mm Hg vs 100.34 [1.02] mm Hg; DBP, 64.7 [1.23] mm Hg vs 59.76 [0.91] mm Hg). No differences in the SBP and DBP were found between children who were overweight or obese whose mothers received DHA and children who were not overweight or obese. In the mixed model analysis, the child's age at blood pressure measurement and the maternal prepregnancy body mass index were the only other statistically significant variables (child age, SBP: F = 7.385; P = .001; DBP: F = 7.39; P = .001; prepregnancy BMI, SBP: r = 0.284; P = .001; DBP: r = 0.216; P = .01). Maternal docosahexaenoic acid intake during pregnancy appeared to mitigate the association between childhood overweight condition or obesity and blood pressure. ClinicalTrials.gov Identifier: NCT02487771.

Importance of pancreatic exocrine dysfunction in patients with type 2 diabetes: A randomized crossover study

BackgroundLevels of faecal elastase-1 (FE-1), a marker of exocrine pancreatic function, are lower in patients with type 2 diabetes than without diabetes. We aimed to investigate the association between FE-1 and nutritional status, gastrointestinal symptoms, and lipid absorption. MethodsThis randomized, open-label, crossover study included 315 patients with type 2 diabetes aged 18–70 years treated with oral antidiabetics, with HbA1c 6.5–9.0% and BMI 18–40 kg/m2. Assessments included levels of FE-1 and blood biomarkers of nutrition, and Bristol Stool Scale and Gastrointestinal Symptom Rating Scale (GSRS) scores. Plasma exposure of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) after oral administration of free omega-3 carboxylic acids or ethyl esters with breakfast was investigated in patients with low, intermediate, and normal FE-1 levels. ResultsThe prevalence of low and intermediate FE-1 levels was 5.2% and 4.9%, respectively. Bristol Stool Scale scores and mean values of GSRS Diarrhoea and Indigestion domain symptoms were similar across groups, but patients with low FE-1 were heavier and reported lower stool frequency. FE-1 levels correlated positively with plasma levels of amylase, lipase, 25-hydroxy vitamin D, and albumin. Mean EPA + DHA exposure was similarly higher after intake of free vs. esterified omega-3 fatty acids in all FE-1 groups. ConclusionsThe prevalence of low FE-1 (<100 μg/g) as a measure of pancreatic exocrine insufficiency was infrequent in type 2 diabetes. Except for low plasma concentrations of EPA and 25-hydroxy vitamin D, type 2 diabetes patients with low FE-1 had no other signs of malabsorption or gastrointestinal disorders. Plasma levels of EPA and DHA after the intake of esterified versus free EPA and DHA did not correlate with FE-1 levels. Trial registrationClinicalTrials.gov NCT02370537.

Intrauterine DHA exposure and child body composition at 5 y: exploratory analysis of a randomized controlled trial of prenatal DHA supplementation

Observational studies find associations between maternal docosahexaenoic acid (DHA) and greater fat-free mass and lower percentage of body fat, but randomized trials of prenatal DHA supplementation have not found significant intent-to-treat effects on childhood body composition. This study sought to explore associations between intrauterine DHA exposure and body composition and size at 5 y in the offspring of women who participated in a randomized trial of prenatal DHA supplementation (corn and soybean oil placebo or 600 mg/d). At 5 y, body composition was measured by air displacement plethysmography in 154 offspring of women who had participated in the Kansas University DHA Outcomes Study and who had red blood cell (RBC) phospholipid (PL) fatty acids assessed at enrollment and delivery. We used linear regression models to analyze the relation among 3 indicators of intrauterine DHA exposure-1) intent-to-treat (placebo or DHA), 2) maternal RBC PL DHA status at delivery, and 3) change in maternal DHA (delivery minus enrollment)-and 6 outcomes of interest: 5-y fat mass, fat-free mass, percentage of body fat, height, weight, and body mass index z score. Change in maternal RBC PL DHA correlated with higher fat-free mass (r = 0.21, P = 0.0088); the association was unchanged after adjustment for maternal, perinatal, and childhood dietary factors. Intent-to-treat and DHA status at delivery showed positive trends with fat-free mass that were not statistically significant. There was no evidence relating intrauterine DHA exposure to any other body composition measure. Change in maternal DHA status during pregnancy was related to higher offspring 5-y fat-free mass. The other 2 indicators of intrauterine exposure to DHA suggested a trend for higher offspring 5-y fat-free mass. Our findings agree with an earlier observational study from the United Kingdom. This trial was registered at clinicaltrials.gov as NCT00266825.

Involvement of apoptotic pathways in docosahexaenoic acid-induced benefit in prostate cancer: Pathway-focused gene expression analysis using RT2 Profile PCR Array System

BackgroundPresent study aimed to better understand the potential apoptotic pathways that involved in docosahexaenoic acid (DHA)-induced apoptosis of prostate cancer cells.MethodsHuman prostate cancer DU145 cells were treated with different concentrations of fish oil, omega-3 PUFA (DHA, and Eicosapentaenoic acid, EPA), or omega-6 PUFA (Arachidonic acid, AA). Cell viability and apoptosis were evaluated by MTT assay and Hoechst staining. Pathway-focused gene expression profiling of DU145 cells was analyzed with the RT2 Profile PCR Array System. The results were verified by real time quantitative polymerase chain reaction (RT-qPCR).ResultsAA exposure showed no obvious effect on viability of DU145 cells. However, exposure with fish oil, EPA, or DHA for 24 h significantly affected cell viability. The growth inhibition of DHA was more pronounced than that of EPA and showed a time-dependent increase. DHA exposure caused typical apoptotic characteristics. Ten genes were more expressed, while 5 genes were less expressed following DHA exposure. RT-qPCR confirmed the time dependent effect of DHA on the expression of these differentially expressed genes. KEGG pathway analysis showed that DHA may induce the apoptosis of cancer cells preferentially through mediating P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways.ConclusionOur study demonstrated the beneficial action of DHA on human prostate carcinoma cell line DU145. The pro-apoptotic effect of DHA on DU145 cells may involve mediation various pathways, especially P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways. Molecular mechanisms of DHA on apoptosis of cancer cells still need to be further clarified.

EPA and DHA exposure alters the inflammatory response but not the surface expression of Toll-like receptor 4 in macrophages.

Dietary intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and their respective enrichment in cell membranes have been negatively associated with atherosclerotic lesion development. This effect may be mediated, in part, by dampened inflammatory response of macrophages triggered by toll-like receptor 4 (TLR4) activation. This study investigated the influence of membrane fatty acid profile on TLR4-mediated inflammation in RAW 264.7 macrophages. Cells pretreated with myristic acid (MA), EPA, DHA or vehicle control for 24 h were stimulated with ultra-pure LPS, a specific TLR4 agonist, for 6 or 24 h, corresponding to early and late stages of TNFα and IL-6 protein induction. Treatment significantly increased cell membrane MA, EPA, and DHA by 4.5-, 20.6-, and 8.9-fold, respectively. MA significantly increased IL-6 secretion 6 h post-exposure to the fatty acid, but did not change TNFα secretion in response to any other treatment condition. EPA and DHA significantly reduced TNFα secretion by 36 and 41 %, respectively, in cells stimulated for 24 h but not 6 h. In contrast, EPA and DHA significantly reduced IL-6 secretion at both 6 h (67 and 72%, respectively) and 24 h (69 and 72%, respectively). MA or DHA treatment had no significant effect compared to vehicle on factors influencing cellular LPS recognition, including LPS-cell association, and cell surface expression of TLR4, TLR4-MD2 complex, and CD14. These data suggest that membrane fatty acid profiles influence the TLR4-mediated inflammatory response in macrophages, via mechanisms that occur downstream of TLR4 receptor activation.

FADS single-nucleotide polymorphisms are associated with behavioral outcomes in children, and the effect varies between sexes and is dependent on PPAR genotype , ,

Docosahexaenoic acid (DHA), supplied by the diet or endogenous biosynthesis from α-linolenic acid, accretes during the perinatal brain growth spurt. Results regarding a potential programming effect on cognitive function and behavior in humans are inconclusive. Here we aimed to investigate whether behavioral outcomes in childhood were associated with FADS tag-single-nucleotide polymorphisms (SNPs) previously found to have opposing effects on infant erythrocyte DHA. At 36 mo, we assessed psychomotor development with the third edition of the Ages & Stages Questionnaire (n = 256) and physical activity by accelerometry (n = 231) in children from the SKOT [Småbørns Kost Og Trivsel (Diet and Thriving in Young Children)] cohort. Blood samples were taken to determine erythrocyte DHA (n = 200), FADS tag-SNPs (n = 255), and PPARG-Pro12Ala (n = 255). All outcomes were analyzed in models, including all 3 SNPs, SNP-sex interactions, erythrocyte DHA at 36 mo, and covariates. As previously shown, the minor allele carriers of the FADS SNP rs1535 had increased erythrocyte DHA at 9 mo, whereas DHA decreased in minor allele carriers of rs174448 and rs174575 (effect size around 0.5 percentage points per allele). No overall effects were observed for any of the FADS SNPs on the outcomes reported here, but FADS SNP-sex interactions were found for a number of DHA-increasing FADS alleles on both communication and problem solving (P = 0.005 and 0.013). DHA-increasing FADS alleles resulted in reduced scores in girls and improved abilities in boys, with an effect size of ∼1 score-point/allele. No associations were found between current erythrocyte DHA and any of the behavioral outcomes. The P value for the triple interaction between DHA-increasing FADS alleles, PPARG, and sex for communication was 0.051, and subsequent analyses showed the FADS-sex interaction only in PPARG minor allele carriers (n = 70). Furthermore, FADS-PPARG interactions were seen for problem solving in boys and for fine motor skills in girls. FADS SNPs seem to have a sex-specific, possibly peroxisome proliferator-activated receptor-mediated effect on behavior in children, indicating a programming effect of early DHA exposure.

Molecular investigation and diagnosis of three genetic diseases in Arabian horses from Middle East and North American populations

Maternal docosahexaenoic acid (DHA) status has been shown to influence cognitive development in infants and children. Improvements in learning ability might enhance trainability of horses, which could lead to better athletic performance and decreased behavioral problems. To assess the cognitive benefits of early exposure to DHA, offspring of mares fed a fat supplement containing an algae source of DHA (n¼10) or a placebo fat supplement (CON, n¼10) from d 250 of gestation through 74 d of lactation were evaluated. Using operant conditioning, foals had been taught to touch a target object with their nose upon verbal command at 2 mo of age. This study evaluated memory recall of previous training, as well as performance on new, more complex tasks using the same target training technique in weaned foals (meanSEM, 185 � 2 d of age). We hypothesized that weanlings exposed to DHA would have better memory recall and progress further and with greater ease through a series of new tasks than weanlings exposed to CON. With researchers blinded to treatment, weanling response times, performance scores, and the number of cues needed to successfully complete each behavior were recorded during 11 training sessions conducted over 6 d. Data were analyzed using a two-tail t-test or zero inflated Poisson model. Memory recall of previous training was evaluated during the first 2 sessions by repeating the original targeting behaviors. Based on performance scores, all foals demonstrated recall of previous training (P< 0.0001). Mean response time during session 1 was slower (P ¼ 0.031) than session 2, indicating optimal recall may not be achieved when first re-exposed. Memory recall was not affected by DHA exposure or sex of the foal. New behavior training (sessions 3-11) was comprised of tar- geting tasks that progressed in difficulty, including walking from point A to B, performing target behaviors in an open environment, walking over novel objects (black stall mat and wooden platform), and loading into a live- stock-hauling trailer. Overall, weanlings exposed to DHA were more likely to score lower (P ¼ 0.016) than CON weanlings on new training tasks. DHA weanlings scored lower (P ¼ 0.013) and required more cues (P ¼ 0.037) to successfully complete the platform task compared to CON weanlings, whereas performance on all other individual tasks was unaffected by maternal diet. When performance on the platform was excluded from analysis, DHA wean-

Effect of early exposure to maternal docosahexaenoic acid on memory and cognition in weaned foals

Effect of early exposure to maternal docosahexaenoic acid on memory and cognition in weaned foals

Abstract 2867: Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling in human cancer cells

Abstract Although Omega 3-polyunsatuarated fatty acids (Omega 3-PUFAs) induce cytotoxicity in several cancer cell lines, the exact mechanisms are not identified yet. In this study, we showed that autophagy is involved in the Omega 3-PUFAs-induced cytotoxicity in cancer cells. Autophagy is characterized by the sequestration of cytoplasmic material within autophagosomes for bulk degradation by lysosomes. We found that docosahexaenoic acid (DHA), an Omega 3-PUFA, induced not only apoptosis but also autophagy in cancer cells. Autophagy was detected after DHA exposure as indicated by induction of LC3 expression, and formation of autophagic vacuolization. We observed that the DHA-induced autophagy was accompanied by loss of p53. Inhibition of p53 by Pifithrin-α or microRNA-p53 significantly increased the DHA-induced autophagy, suggesting that the DHA-induced autophagy is mediated by downregulation of p53. Further experiments showed that the mechanism of the DHA-induced autophagy associated with p53 attenuation, involved an increase in the active form of AMPK which attenuated the mTOR activity as revealed by p27 sequestration. In addition, compelling evidence for the interplay between autophagy and apoptotic cell death induced by DHA is supported by the findings that autophagy inhibition partially decreased the DHA-induced apoptotic cell death and further autophagy induction by p53 inhibitor enhanced apoptosis in response to treatment with DHA in cancer cells. Our results demonstrate that autophagy may be related to the DHA-induced cytotoxicity in cancer cells. [This work was supported by basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (E00026 and R13-2007-020-01000-0]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2867. doi:10.1158/1538-7445.AM2011-2867

Anti-inflammatory properties of docosahexaenoic acid (DHA) in bone marrow-derived dendritic cells (98.11)

Abstract Omega 3 fatty acids have anti-inflammatory effects in chronic inflammatory and autoimmune diseases. Previous studies showed that dendritic cells (DC) differentiated in the presence of DHA express reduced costimulatory molecules and secrete lower levels of cytokines. We report that 24h exposure to DHA of immature DC prevents LPS-induced upregulation of MHCII, CD40, CD80 and CD86. Immature DC are characterized by CCR5 expression and migration to proinflammatory chemokines, whereas mature DC express CCR7 and migrate to lymph nodes in response to CCL19/21. DHA exposure kept CCR5 expression at high level, and prevented CCR7 expression induced by LPS. DHA-treated DC migrated poorly towards CCL19. Proinflammatory cytokines including IL-12p70, IL-23, IL-6 and TNFα produced by mature DC were inhibited by DHA. We found that DHA inhibited IL-12p70 and IL-23 production induced by ligands for TLR4 and 2, and also by ligands for the intracellular TLR3 and 9. In agreement with previous reports showing that omega 3 fatty acids bind to PPARγ, we found that DHA induced nuclear PPARγ translocation, and that a PPARγ antagonist partially reversed the inhibitory effect of DHA on IL-12p70. In conclusion, our studies indicate that exposure to DHA maintains already differentiated DC in an immature state and prevents expression of proinflammatory cytokines and chemokines. Support NIH 2RO1AI052306.

Docosahexaenoic acid supplementation in pregnancy and lactation

The goal of the Experimental Biology symposium on maternal supplementation was to review all available lines of evidence, delineate unanswered questions, and develop, if it seemed reasonable, a research agenda to determine whether maternal supplementation with specific nutrients might be beneficial. In the case of maternal docosahexaenoic acid (DHA) status, the topic addressed in this article, few clinical studies show benefits of maternal DHA supplementation during pregnancy or lactation for the infant or child. However, quite a large number of observational studies link higher intrauterine DHA exposure to a number of positive developmental outcomes. This article reviews the factors known to contribute to DHA status of women and their offspring during the reproductive cycle, relates maternal DHA status to that of the developing fetus and newborn, and reviews the evidence for functional differences in behavior related to DHA status, including the available evidence related to DHA supplementation of women pregnant and lactating and their offspring. Other outcomes for infants and children and for women themselves appear plausible and are also addressed as part of a research agenda for future work.

Effects of arachidonic acid and docosahexaenoic acid on differentiation and mineralization of MC3T3‐E1 osteoblast‐like cells

Osteoblasts in culture can differentiate into mature mineralizing osteoblasts when stimulated with osteogenic agents. Clinical trials and in vivo animal studies suggest that specific polyunsaturated fatty acids (PUFAs) may benefit bone health. The aim of this study was to investigate whether arachidonic acid (AA) and docosahexaenoic acid (DHA) affect osteogenesis in osteoblasts and the transdifferentiation into adipocytes. Results from this study show that long-term exposure to AA inhibited alkaline phosphatase (ALP) activity in these cells, which might be prostaglandin E(2) (PGE(2))-mediated. DHA exposure also inhibited ALP activity which was evident after both short- and long-term exposures. The mechanism whereby DHA inhibits ALP activity is not clear and needs to be investigated. Although long-term exposure to PUFAs inhibited ALP activity, the mineralizing properties of these cells were not compromised. Furthermore, PUFA exposure did not induce adipocyte-like features in these cells as evidenced by the lack of cytoplasmic triacylglycerol accummulation. More research is required to elucidate the cellular mechanisms of action of PUFAs on bone.

Docosahexaenoic acid may act as a neuroprotector for methylmercury-induced neurotoxicity in primary neural cell cultures

The ability of docosahexaenoic acid (DHA) to modulate methylmercury (MeHg)-induced neurotoxicity was investigated in primary astrocytes and neurons from the cerebellum. Gas chromatography measurements indicated increased DHA content in both cell types after 24 h supplementation. After individual or combined treatment with MeHg (10 μM) and DHA (30 and 90 μM), the cell-associated MeHg measurements were done using 14C-labelled MeHg. In addition, mitochondrial activity was evaluated by MTT reduction, glutathione (GSH) content was measured with the fluorescent indicator monochlorobimane (MCB) and reactive oxygen species (ROS) were detected with the fluorescent indicator—chloro methyl derivative of di-chloro di-hydro fluorescein diacetate (CMH 2DCFDA). For all the tested treatments, i.e. DHA, MeHg or DHA + MeHg treatment, the neurons differed significantly ( p < 0.001) from astrocytes exhibiting increased ROS production and decreased MTT activity. After MeHg and 30 μM DHA treatment there were no changes in MTT or GSH content but significant decrease ( p < 0.001) in ROS was observed in both the cell types when compared to MeHg alone. The cell-associated MeHg measurements indicated reduced MeHg-accumulation in both cell types ( p < 0.05) upon 30 μM DHA exposure. Taken together, this study, for the first time establishes that DHA pretreatment effectively reduces cell-associated MeHg and prooxidant response from MeHg in both cerebellar astrocytes and neurons and thus supports the hypothesis that fish-derived nutrients offer possible neuroprotection from MeHg.

Role of Docosahexaenoic Acid in Modulating Methylmercury-Induced Neurotoxicity

The effect of docosahexaenoic acid (DHA) in modulating methylmercury (MeHg)-induced neurotoxicity was investigated in C6-glial and B35-neuronal cell lines. Gas chromatography measurements indicated increased DHA content in both the cell lines after 24 h supplementation. Mitochondrial activity evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) reduction indicated that 10 microM MeHg treatment for 50 min led to a significant (p < 0.001) and similar decrease in MTT activity in both the cell lines. However, DHA pretreatment led to more pronounced depletion (p < 0.05) in the MTT activity in C6 cells as compared to B35 cells. The depletion of glutathione (GSH) content measured with the fluorescent indicator monochlorobimane was more apparent (p < 0.001) in C6 cells treated with DHA and MeHg. The amount of reactive oxygen species (ROS) detected with the fluorescent indicator -- chloromethyl derivative of dichloro dihydro fluorescein diacetate (CMH(2)DCFDA) -- indicated a fourfold increase in C6 cells (p < 0.001) as compared to twofold increase in B35 cells (p < 0.001) upon DHA and MeHg exposure. However, the cell-associated MeHg measurement using (14)C-labeled MeHg indicated a decrease (p < 0.05) in MeHg accumulation upon DHA exposure in both the cell lines. These findings provide experimental evidence that although pretreatment with DHA reduces cell-associated MeHg, it causes an increased ROS (p < 0.001) and GSH depletion (p < 0.05) in C6 cells.

Oleic acid and docosahexaenoic acid cause an increase in the paracellular absorption of hydrophilic compounds in an experimental model of human absorptive enterocytes

Surface active compounds present in food possibly have the ability to enhance the absorption of water soluble toxic agents. Therefore, we investigated whether fatty acids such as oleic acid and docosahexaenoic acid (DHA), both commonly present in food, negatively affect the integrity of tight junctions (TJ) in the intestinal epithelium and thereby increase the absorption of poorly absorbed hydrophilic substances. Caco-2 cells, which are derived from human absorptive enterocytes, were grown on permeable filters for 20–25 days. Differentiated cell monolayers were apically exposed for 90min to mannitol in emulsions of oleic acid (5, 15 or 30mM) or DHA (5, 15 or 30mM) in an experimental medium with or without Ca2+ and Mg2+. Absorption of 14C-mannitol increased and trans-epithelial electrical resistance (TEER) decreased in cell monolayers exposed to oleic acid and DHA, compared to controls. Cytotoxicity, measured as leakage of LDH, was higher in groups exposed to 30mM oleic acid and all concentrations of DHA. Morphology of the cell monolayers was studied by using fluorescence microscopy. Exposure of cell monolayers to 5mM DHA for 90min resulted in a profound alteration of the cell–cell contacts as detected by staining the cells for β-catenin. Oleic acid (30mM) treatment also induced dissolution of the cell–cell contacts but the effect was not as pronounced as with DHA. Cell monolayers were also exposed for 180min to 250nM cadmium (Cd) in emulsions of oleic acid (5 or 30mM) or DHA (1 or 5mM), in an experimental medium with Ca2+ and Mg2+. Retention of Cd in Caco-2 cells was higher after exposure to 5mM oleic acid but lower after exposure to 30mM oleic acid and DHA. Absorption of Cd through the monolayers increased after DHA exposure but not after exposure to oleic acid. Our results indicate that fatty acids may compromise the integrity of the intestinal epithelium and that certain lipids in food may enhance the paracellular absorption of poorly absorbed hydrophilic substances.

A probabilistic model for simultaneous exposure to multiple compounds from food and its use for risk–benefit assessment

A model is presented which allows to quantify the simultaneous distribution of the exposure to two compounds, for example a health-risk and a health promoting compound. The model considers the total dietary intake, and can be used as a first step to study the effects on the balance between risks and benefits following changes in the consumption pattern. The exposure is modelled separately for intake probabilities using a betabinomial model, and for intake amounts using a lognormal model, and these parts are afterwards integrated by Monte Carlo simulation. The model is illustrated using the risk–benefit case of dioxins and the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). High concentrations of both the health adverse compounds and the health promoting compounds are simultaneously present in fatty fish. Calculated exposures were compared with intake limits: the adequate intake for EPA + DHA and the tolerable daily intake (TDI) for dioxins. We estimate the probability that dioxin exposure is below TDI, the probability that EPA + DHA exposure is above the adequate intake, and the probability that both conditions occur simultaneously. We also model the dependence of these probabilities on age. In the studied population the exposure to both compounds is almost completely below the limits. A scenario study in which meat consumption was replaced by fatty fish consumption shows an increase in the fraction of the population with the recommended intake of EPA + DHA, however also the fraction of the population exceeding the TDI for dioxins is increased. For the example scenario the optimal amount of fatty fish consumption is derived.

N−3 Fatty acids and cognitive and visual acuity development: methodologic and conceptual considerations

Several randomized clinical studies in infants born preterm and at term have explored the effects on visual acuity development of postnatal supplementation with various sources of docosahexaenoic acid (DHA). Higher visual acuity after DHA supplementation is a consistent finding in infants born preterm. For infants born at term, the results are less consistent and are better explained by differences in sensitivity of the visual acuity test (electrophysiologic tests being more sensitive than subjective tests) or by differences in the amount of DHA included in the experimental formula. Differences in the sensitivity of the test may also be relevant in discussions of whether the effects of DHA on visual acuity are transient or persistent. A smaller number of studies have attempted to study the effects of DHA on cognitive development. The major focus of this article is to review the types of methods that have been used to evaluate the effects of DHA on cognition and to provide the rationale for measures that are a better conceptual fit. Research is needed (1) to probe the effects of variable DHA exposure on infant and child development, (2) to measure outcomes that better relate to preschool and school-age cognitive function, and (3) to reinforce, and in some cases demonstrate, links between specific infant and preschool measures of cognitive development. We strongly encourage collaborations with developmental cognitive neuroscientists to facilitate these research goals.