Crystalline silica-induced pulmonary inflammation and autoimmunity in mature adult NZBW/f1 mice: age-related sensitivity and impact of omega-3 fatty acid intervention.

OBJECTIVE: Occupational exposure to respirable crystalline silica (cSiO2) has been linked to lupus development. Previous studies in young lupus-prone mice revealed that intranasal cSiO2 exposure triggered autoimmunity, preventable with docosahexaenoic acid (DHA). This study explores cSiO2 and DHA effects in mature lupus-prone adult mice, more representative of cSiO2-exposed worker age. METHODS: Female NZBWF1 mice (14-week old) were fed control (CON) or DHA-supplemented diets. After two weeks, mice were intranasally instilled saline (VEH) or 1 mg cSiO2 weekly for four weeks. Cohorts were then analyzed 1- and 5-weeks postinstillation for lung inflammation, cell counts, chemokines, histopathology, B- and T-cell infiltration, autoantibodies, and gene signatures, with results correlated to autoimmune glomerulonephritis onset. RESULTS: VEH/CON mice showed no pathology. cSiO2/CON mice displayed significant ectopic lymphoid tissue formation in lungs at 1 week, increasing by 5 weeks. cSiO2/CON lungs exhibited elevated cellularity, chemokines, CD3+ T-cells, CD45R + B-cells, IgG + plasma cells, gene expression, IgG autoantibodies, and glomerular hypertrophy. DHA supplementation mitigated all these effects. DISCUSSION: The mature adult NZBWF1 mouse used here represents a life-stage coincident with immunological tolerance breach and one that more appropriately represents the age (20-30 yr) of cSiO2-exposed workers. cSiO2-induced robust pulmonary inflammation, autoantibody responses, and glomerulonephritis in mature adult mice, surpassing effects observed previously in young adults. DHA at a human-equivalent dosage effectively countered cSiO2-induced inflammation/autoimmunity in mature mice, mirroring protective effects in young mice. CONCLUSION: These results highlight life-stage significance in this preclinical lupus model and underscore omega-3 fatty acids' therapeutic potential against toxicant-triggered autoimmune responses.

Safety assessment of EPA+DHA canola oil by fatty acid profile comparison to various edible oils and fat-containing foods and a 28-day repeated dose toxicity study in rats.

The omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are recognized for their health-promoting qualities. Marine fish and fish oil currently provide the main sources of EPA and DHA for human consumption. An alternative plant-based source of EPA and DHA is provided by EPA + DHA canola event LBFLFK (LBFLFK). A comparative analysis and a 28-day toxicity study assessed the safety of LBFLFK refined, bleached, and deodorized (RBD) oil. Thirty-one different commercially-obtained fat and oil samples were tested, and principal component analysis showed that the overall fatty acid profile of LBFLFK RBD oil was most similar to Mortierella alpina oil and salmon flesh. Samples with the fewest differences in the presence or absence of individual fatty acids compared to LBFLFK RBD oil were menhaden oil and some other fish oils. In a 28-day toxicity study, LBFLFK RBD oil was administered by oral gavage to male and female Wistar rats. No signs of toxicity were evident and no adverse effects were noted in clinical observations, clinical pathology, or histopathology. Overall, these studies support the safety of LBFLFK RBD oil as a source of EPA and DHA for human consumption.

Is cytotoxicity a determinant of the different in vitro and in vivo effects of bioactives?

BACKGROUND: Foodstuffs of both plant and animal origin contain a wide range of bioactive compounds. Although human intervention studies are mandatory to assess the health effects of bioactives, the in vitro approach is often used to select the most promising molecules to be studied in vivo. To avoid misleading results, concentration and chemical form, exposure time, and potential cytotoxicity of the tested bioactives should be carefully set prior to any other experiments. METHODS: In this study the possible cytotoxicity of different bioactives (docosahexaenoic acid, propionate, cyanidin-3-O-glucoside, protocatechuic acid), was investigated in HepG2 cells using different methods. Bioactives were supplemented to cells at different concentrations within the physiological range in human blood, alone or in combination, considering two different exposure times. RESULTS: Reported data clearly evidence that in vitro cytotoxicity is tightly related to the exposure time, and it varies among bioactives, which could exert a cytotoxic effect even at a concentration within the in vivo physiological blood concentration range. Furthermore, co-supplementation of different bioactives can increase the cytotoxic effect. CONCLUSIONS: Our results underline the importance of in vitro cytotoxicity screening that should be considered mandatory before performing studies aimed to evaluate the effect of bioactives on other cellular parameters. Although this study is far from the demonstration of a toxic effect of the tested bioactives when administered to humans, it represents a starting point for future research aimed at verifying the existence of a potential hazard due to the wide use of high doses of multiple bioactives.

Antibacterial and antibiofilm activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against periodontopathic bacteria.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two major omega-3 polyunsaturated fatty acids (n-3 PUFAs) with antimicrobial properties. In this study, we evaluated the potential antibacterial and antibiofilm activities of DHA and EPA against two periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). MTT assay showed that DHA and EPA still exhibited no cytotoxicity to human oral tissue cells when the concentration came to 100 µM and 200 µM, respectively. Against P. gingivalis, DHA and EPA showed the same minimum inhibitory concentration (MIC) of 12.5 µM, and a respective minimum bactericidal concentration (MBC) of 12.5 µM and 25 µM. However, the MIC and MBC values of DHA or EPA against F. nucleatum were both greater than 100 µM. For early-stage bacteria, DHA or EPA displayed complete inhibition on the planktonic growth and biofilm formation of P. gingivalis from the lowest concentration of 12.5 µM. And the planktonic growth of F. nucleatum was slightly but not completely inhibited by DHA or EPA even at the concentration of 100 µM, however, the biofilm formation of F. nucleatum at 24 h was significantly restrained by 100 µM EPA. For exponential-phase bacteria, 100 µM DHA or EPA completely killed P. gingivalis and significantly decreased the viable counts of F. nucleatum. Meanwhile, the morphology of P. gingivalis was apparently damaged, and the virulence factor gene expression of P. gingivalis and F. nucleatum was strongly downregulated. Besides, the viability and the thickness of mature P. gingivalis biofilm, together with the viability of mature F. nucleatum biofilm were both significantly decreased in the presence of 100 µM DHA or EPA. In conclusion, DHA and EPA possessed antibacterial activities against planktonic and biofilm forms of periodontal pathogens, which suggested that DHA and EPA might be potentially supplementary therapeutic agents for prevention and treatment of periodontal diseases.

Sub-chronic (13-week) oral toxicity study, preceded by an in utero exposure phase and genotoxicity studies with fish source phosphatidylserine in rats.

The safety of fish phosphatidylserine (PS) conjugated to DHA (InCog™) was examined in a series of toxicology studies as first step to support future use in infants and general population using in vitro genotoxicity tests and in a sub-chronic toxicity study with an in-utero exposure phase. PS is a major lipid in the cell membrane, active in various membrane-mediated processes. PS-DHA, present in human milk, has been suggested to be important for early brain development. Rats were exposed to diets containing 1.5%, 3% or 4.5% InCog or two control diets. Parental (F0) animals were fed throughout mating, gestation and lactation. Subsequently, a subchronic, 13-week study was conducted on the F1 animals followed by 4 weeks of recovery. The genotoxicity tests showed no mutagenicity potential. No significant toxicological findings were found in the F0 rats or the F1 pups. In the 13-weeks study, an increase in the presence of renal minimal-mild multifocal corticomedullary mineralization was noted in nine females of the high-dose group. This change was not associated with any inflammatory or degenerative changes in the kidneys. The no-observed-adverse-effect level (NOAEL) in the present study was placed at 3% in the diet (mid-dose group), equivalent to an overall intake of at least 2.1 g InCog/kg bw/day in the F1 generation.

Dietary long-chain omega-3 fatty acids do not diminish eosinophilic pulmonary inflammation in mice.

Although the effects of fish oil supplements on airway inflammation in asthma have been studied with varying results, the independent effects of the fish oil components, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), administered separately, are untested. Here, we investigated airway inflammation and hyperresponsiveness using a mouse ovalbumin exposure model of asthma assessing the effects of consuming EPA (1.5% wt/wt), DHA (1.5% wt/wt), EPA plus DHA (0.75% each), or a control diet with no added omega-3 polyunsaturated fatty acids. Consuming these diets for 6 weeks resulted in erythrocyte membrane EPA contents (molar %) of 9.0 (± 0.6), 3.2 (± 0.2), 6.8 (± 0.5), and 0.01 (± 0.0)%; DHA contents were 6.8 (± 0.1), 15.6 (± 0.5), 12.3 (± 0.3), and 3.8 (± 0.2)%, respectively. The DHA group had the highest bronchoalveolar lavage (BAL) fluid eosinophil and IL-6 levels (P < 0.05). Similar trends were seen for macrophages, IL-4, and IL-13, whereas TNF-α was lower in omega-3 polyunsaturated fatty acid groups than the control (P < 0.05). The DHA group also had the highest airway resistance, which differed significantly from the EPA plus DHA group (P < 0.05), which had the lowest. Oxylipins were measured in plasma and BAL fluid, with DHA and EPA suppressing arachidonic acid-derived oxylipin production. DHA-derived oxylipins from the cytochrome P450 and 15-lipoxygenase pathways correlated significantly with BAL eosinophil levels. The proinflammatory effects of DHA suggest that the adverse effects of individual fatty acid formulations should be thoroughly considered before any use as therapeutic agents in asthma.

DHA sensitizes FaO cells to tert-BHP-induced oxidative effects. Protective role of EGCG.

The excessive production of reactive oxygen species has been implicated in several pathologies, such as atherosclerosis, obesity, hypertension and insulin resistance. Docosahexaenoic acid (DHA) may protect against the above mentioned diseases, but paradoxically the main DHA treated pathologies are also associated with increased ROS levels. Therefore, the aim of this study was to explore if in vitro DHA supplementation may increase the sensitivity of cells to tert-BHP induced oxidative stress, and if the green tea polyphenol epigallocatechin-3-gallate (EGCG) is able to correct such detrimental effect. We found that DHA-enriched cells exacerbate ROS generation, decrease cell viability and increase Nrf2 nuclear translocation and HO-1 expression. Interestingly, cellular EGCG is able to counteract oxidative damage from either tert-BHP or DHA-enriched cells. In consequence, our results suggest that in a ROS enriched environment DHA could not always be beneficial for cells and can be considered a double-edged sword in terms of its benefits vs. risks. In this sense, our results propose that the supplementation with potent antioxidant molecules could be an appropriate strategy to reduce the risks related with the DHA supplementation in an oxidative stress-associated condition.

Toxicologic evaluations of DHA-rich algal oil in rats: developmental toxicity study and 3-month dietary toxicity study with an in utero exposure phase.

DHA-rich algal oil ONC-T18, tested for subchronic, reproductive, and developmental toxicity in the rat, did not produce any significant toxicologic manifestations. Based on the absence of maternal or developmental toxicity at any dosage level, a dosage level of 2000 mg/kg/day was considered to be the no observed adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when DHA-rich algal oil was administered orally by gavage to pregnant Crl:CD(SD) rats during gestation days 6-19. In a dietary combined one-generation/90-day reproductive toxicity study in rats, the NOAEL for F0 male and female and F1 male systemic toxicity was considered to be 50,000 ppm (highest concentration administered) and 25,000 ppm for F1 female systemic toxicity (higher mean body weight, body weight gain, and food consumption). F0 reproductive performance values, estrous cycle length, gestation length, or the process of parturition, and the numbers of former implantation sites and unaccounted-for sites were unaffected by algal oil exposure. Postnatal survival and developmental parameters in the F1 generation were unaffected by algal oil exposure at all dietary concentrations. There were no neurotoxic effects noted at any algal oil exposure level. The results support the safety of DHA-rich algal oil for its proposed use in food.

Safety evaluation of DHA-rich Algal Oil from Schizochytrium sp.

The safety of DHA-rich Algal Oil from Schizochytrium sp. containing 40-45 wt% DHA and up to 10 wt% EPA was evaluated by testing for gene mutations, clastogenicity and aneugenicity, and in a subchronic 90-day Sprague-Dawley rat dietary study with in utero exposure, followed by a 4-week recovery phase. The results of all genotoxicity tests were negative. In the 90-day study, DHA-rich Algal Oil was administered at dietary levels of 0.5, 1.5, and 5 wt% along with two control diets: a standard low-fat basal diet and a basal diet supplemented with 5 wt% of concentrated Fish Oil. There were no treatment-related effects of DHA-rich Algal Oil on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, coagulation, or urinalysis. Increases in absolute and relative weights of the liver, kidney, spleen and adrenals (adrenals and spleen with histological correlates) were observed in both the Fish Oil- and the high-dose of DHA-rich Algal Oil-treated females and were not considered to be adverse. The no observed adverse effect level (NOAEL) for DHA-rich Algal Oil under the conditions of this study was 5 wt% in the diet, equivalent to an overall average DHA-rich Algal Oil intake of 4260 mg/kg bw/day for male and female rats.

Preclinical safety evaluation in rats using a highly purified ethyl ester of algal-docosahexaenoic acid.

Preclinical studies have shown that docosahexaenoic acid (DHA) derived from microalgae (DHASCO) is neither mutagenic nor toxic in acute, subchronic or developmental tests. DHASCO, triglyceride oil from the fermentation of Crypthecodinium cohnii, contains 40-50% (400-500 mg/g) of DHA by weight. Martek Biosciences Corporation has developed a concentrated ethyl ester of DHA (900 mg/g) from DHASCO (MATK-90). A 90-day subchronic safety study with a one-month recovery period using Sprague-Dawley rats included clinical observations, ophthalmic examination, hematology, clinical chemistry, toxicokinetic evaluation, and pathological assessments. Effects of MATK-90 were compared with those produced from DHASCO and control (corn oil). Doses of MATK-90 (1.3, 2.5 and 5.0 g/kg/day) and DHASCO (5.0 g/kg/day=2g of DHA) were administered once-daily by oral gavage at a volume of 10 mL/kg. The corn oil was also administered by oral gavage (10 mL/kg/day). There were no treatment-related adverse effects in any of the parameters measured at doses of

Toxicology and safety of DHA.

Docosahexaenoic acid (DHA) is a long-chain polyunsaturated fatty acid with activities in both infants and adults. The objective of the current work was to evaluate the published literature concerning the toxicological assessment of DHA-rich oils in animals and the safety profile of DHA consumption in humans. Structured literature searches concerning DHA toxicology and DHA effects on platelet function, lipid levels, oxidative potential, glycemic control, and immune function were conducted. The toxicological profile of DHA derived from single-cell organisms demonstrates that these oils are safe in rats (up to a consumption level of 3290 mg/kg body weight/d) in 90-d toxicology evaluations, as well as in reproductive and developmental toxicology studies. The maximum DHA level in human breast milk exceeds 1% of total fatty acids in high-fish-consuming populations. Consumption of DHA-rich human milk as sole source of nutrition provides approximately 315 mg/d in infants 1-6 months of age, and appears to be a safe level of intake. DHA supplementation studies in adults have employed doses ranging from less than 1 to 7.5 g/d, and have not resulted in any consistent adverse responses in platelet function, lipid levels, in vivo oxidation parameters, glycemic control, or immune function. In conclusion, DHA consumption does not result in consistent adverse events in infants or adults. Safe intake levels may be modeled on DHA intake from human milk in infants, and may be at least as high as the upper doses studied in adults.

Docosahexaenoic and eicosapentaenoic acids increase neuronal death in response to HuPrP82-146 and Abeta 1-42.

Dietary supplements containing polyunsaturated fatty acids (PUFA) are frequently taken for their perceived health benefits including a possible reduction in cognitive decline in the elderly. Here we report that pre-treatment with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) significantly reduced the survival of cortical or cerebellar neurons incubated with HuPrP82-146, a peptide derived from the prion protein, or with Abeta 1-42, a peptide found in Alzheimer's disease. Treatment with DHA or EPA reduced the free cholesterol content of neuronal membranes. This did not affect the amount of FITC-HuPrP82-146 ingested by neurons, but increased the kinetics of incorporation. In untreated neurons, FITC-HuPrP82-146 migrated to caveolin-1 containing lipid rafts. The addition of HuPrP82-146 also triggered the migration of cytoplasmic phospholipase A2 (cPLA2) into caveolin-1 containing rafts, and increased prostaglandin E2 production. Activation of cPLA2 and prostaglandin E2 production were both increased in neurons pre-treated with DHA. These results are consistent with DHA or EPA altering cell membranes resulting in increased amounts of HuPrP82-146 localising to caveolin-1 containing rafts, increased activation of cPLA2, prostaglandin E2 production, caspase-3 activity and reduced neuronal survival. Such observations raise the possibility that some PUFA supplements may accelerate neuronal loss in the terminal stages of prion or Alzheimer's diseases.

One-generation reproductive toxicity study of DHA-rich oil in rats.

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are natural constituents of the human diet. DHA-algal oil is produced through the use of the marine protist, Ulkenia sp. The reproductive toxicity of DHA-algal oil was assessed in a one-generation study. Rats were provided diets containing DHA-algal oil at concentrations of 1.5, 3.0, or 7.5%, and the control group received a diet containing 7.5% corn oil. Males and females were treated for 10 weeks prior to mating and during mating. Females continued to receive test diets during gestation and lactation. In parental animals, clinical observations, mortality, fertility, and reproductive performance were unaffected by treatment. Differences in food consumption, body weight, and liver weight in the treated groups were not considered to be due to an adverse effect of DHA-algal oil. Spleen weight increases in treated animals were associated with extramedullary hematopoiesis. Yellow discoloration of abdominal adipose tissue was observed in rats from the high-dose group, and histological examination revealed steatitis in all treated parental groups. Exposure to DHA-algal oil did not influence the physical development of F(1) animals. These results demonstrate that DHA-algal oil at dietary concentrations of up to 7.5% in rats does not affect reproductive capacity or pup development.

Subchronic (13-week) oral toxicity study, preceded by an in utero exposure phase, with arachidonate-enriched triglyceride oil (SUNTGA40S) in rats.

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA) and docosahexaenoic acid (DHA) are natural constituents found in human milk, fish oil or egg yolk. Until recently, infant formulas, though providing the essential fatty acid precursors for these PUFAs, did not contain preformed ARA or DHA. In this study the safety of SUNTGA40S as source of ARA, not only for use in infant formulas but also for nutritional products or food supplements, was evaluated in a subchronic study in Wistar rats, preceded by a 4-week pretreatment period of parental (F(0)) rats and exposure of the F(0) dams throughout mating, gestation and lactation. SUNTGA40S was administered at dietary levels of 0.5%, 1.5% and 5% (wt/wt) adjusted with corn oil to 5.76% added fat. An additional group received 3.65% (wt/wt) SUNTGA40S in conjunction with 2.11% (wt/wt) high DHA Tuna oil, providing an ARA:DHA ratio of 2.7:1. High-fat and low-fat controls received basal diet with or without 5.76% corn-oil supplement. The content, stability and homogeneous distribution of the test substances in the diet were confirmed under study conditions. The administration of SUNTGA40S, with or without DHA oil, did not affect health, growth, fertility or reproductive performance of the parental rats, nor pup characteristics (condition, weight gain, viability, number per litter or sex ratio). In the subchronic study with the offspring (F(1)) rats, no significant differences were found in condition, neurobehavioural observations, ophthalmoscopy, growth, urinalysis or macroscopic and microscopic findings between the test groups and the low-fat or the high-fat controls. In males of the 5% SUNTGA40S and the SUNTGA40S/DHA group, red blood cell counts, haemoglobin concentration and packed cell volume were lower and reticulocytes were slightly higher than in the high-fat and low-fat control groups. Cholesterol, triglycerides and phospholipids in plasma were lower than in the high-fat controls in both sexes in the 5% SUNTGA40S and the SUNTGA40S/DHA group and (for triglycerides only) in the 1.5% SUNTGA group. Due to the administration of extra dietary fat, food intake and prothrombin time (males only) were lower and alkaline phosphatase activity was higher in all the high-fat groups, including the corn-oil controls, as compared to the low-fat controls. The weight of the spleen was higher in males of the 5% SUNTGA40S and the SUNTGA40S/DHA group compared to both the low-fat and the high-fat controls. The effects noted in this study at high dose levels of SUNTGA40S are consistent with previously reported physiological responses to dietary intake of high PUFA containing oils. The present results provide evidence that SUNTGA40S is a safe source of arachidonic acid. Except during lactation when the intake in dams doubled, 5% Suntga40S in the diet was equivalent to an overall intake of approximately 3g/kg body weight/day in F(0) and F(1) animals.

Effect of arachidonic, eicosapentaenoic and docosahexaenoic acids on the oxidative status of C6 glioma cells.

n-3 polyunsaturated fatty acids (PUFAs) have been described to have beneficial effects on brain development and in the prevention and treatment of brain damage. C6 glioma cells were incubated with 100 microM of either C20:4n-6 (ARA), or C20:5n-3 (EPA), or C22:6n-3 (DHA) for different time periods to assess whether these acids altered the cellular oxidative state. The ARA and EPA were promptly metabolised to C22:4n-6 and C22:5n-3, respectively, whereas DHA treatment simply increased the amount of DHA in the cells. Cell viability was not affected by ARA, while a cytotoxic effect was observed 72 h after n-3 PUFAs supplementation. The levels of reactive oxygen species and thiobarbituric acid-reactive substances were significantly higher in DHA-treated cells than in EPA- and ARA-treated groups. This modification in the oxidative cellular status was also highlighted by a significant increase in catalase activity and a decrease in glutathione content in DHA-supplemented cells. Glucose-6-phosphate dehydrogenase activity, an enzyme involved in redox regulation, and O2*- release were significantly increased both in EPA and DHA groups. The effect of DHA was more severe than that of EPA. No significant changes were observed in the ARA group with respect to untreated cells. These data show that EPA and DHA induce alterations in the oxidative status that could affect the glial function.

A review of the safety of DHA45-oil.

Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), are natural constituents of the human diet; however, dietary intakes of these fatty acids are below recommended values. The main dietary source of DHA is fatty fish, with lesser amounts provided by shellfish, marine mammals, and organ meats. The addition to traditional food products of refined oils produced by marine microalgae represents potential sources of supplemental dietary DHA. DHA45-oil is manufactured through a multi-step fermentation and refining process using a non-toxigenic and non-pathogenic marine protist. Comprising approximately 45% DHA, and lesser concentrations of palmitic acid and docosapentaenoic acid, DHA45-oil is intended for use in foods as a dietary source of DHA. The safety of DHA45-oil was evaluated in various genotoxicity and acute, subchronic, and reproductive toxicity studies. DHA45-oil produced negative results in genotoxicity assays and demonstrated a low acute oral toxicity in mice and rats. Dietary administration of DHA45-oil to rats in subchronic and one-generation reproductive studies produced results consistent with those observed in oral studies using high concentrations of omega-3 PUFAs from fish or other microalgal-derived oils. The results of these studies, as well as those of various published metabolic, toxicological, and clinical studies with DHA-containing oils, support the safety of DHA45-oil as a potential dietary source of DHA.

Safety assessment of DHA-rich microalgae from Schizochytrium sp.

The purpose of this series of studies was to assess the genotoxic potential of docosahexaenoic acid-rich microalgae from Schizochytrium sp. (DRM). DRM contains oil rich in highly unsaturated fatty acids (PUFAs). Docosahexaenoic acid (DHA n-3) is the most abundant PUFA component of the oil ( approximately 29% w/w of total fatty acid content). DHA-rich extracted oil from Schizochytrium sp. is intended for use as a nutritional ingredient in foods. All in vitro assays were conducted with and without mammalian metabolic activation. DRM was not mutagenic in the Ames reverse mutation assay using five different Salmonella histidine auxotroph tester strains. Mouse lymphoma suspension assay methodology was found to be inappropriate for this test material because precipitating test material could not be removed by washing after the intended exposure period and the precipitate interfered with cell counting. The AS52/XPRT assay methodology was not subject to these problems and DRM was tested and found not to be mutagenic in the CHO AS52/XPRT gene mutation assay. DRM was not clastogenic to human peripheral blood lymphocytes in culture. Additionally, DRM did not induce micronucleus formation in mouse bone marrow in vivo further supporting its lack of any chromosomal effects. Overall, the results of this series of mutagenicity assays support the conclusion that DRM does not have any genotoxic potential.

Evaluation of single-cell sources of docosahexaenoic acid and arachidonic acid: 3-month rat oral safety study with an in utero phase.

Owing to the presence of the polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA) and arachidonic acid (ARA) in human milk and their important biological function, several authorities recommend that they be added to infant formulas. This study assessed the safety of an algal oil rich in DHA and a fungal oil rich in ARA, blended to provide a DHA to ARA ratio similar to human milk. The oil blend was incorporated into diets and fed to rats such that they received 3, 11 and 22 times the anticipated infant exposure to DHA and ARA. Low-fat and high-fat control groups received canola oil. Rats received experimental diets over a premating interval and throughout mating, gestation and lactation. Pups born during this period (F1) consumed treatment diets from weaning for 3 months. Physical observations, ophthalmoscopic examinations, body weight, food intake, clinical chemistry, neurobehavioural evaluations and postmortem histopathology of selected tissues were performed. No statistically significant, dose-dependent adverse effects were seen in reproductive performance or fertility, nor in the neonates from birth to weaning. Mid- and high-dose treated F1 animals exhibited increased white cell count, neutrophil count and blood urea nitrogen; increased liver and spleen weights (absolute and relative to body weight) also were observed. There were no corresponding microscopic findings. The clinical pathology and organ weight differences at these treatment levels represent physiological or metabolic responses to the test substance rather than adverse responses. These single-cell oils produced no adverse effects in rats when administered in utero and for 90 days at dietary levels resulting in exposures up to 22 or 66 times higher than those expected in infant formulas when extrapolated on the basis of diet composition (g/100 Cal) or intake (g/kg body weight), respectively.

Dietary docosahexaenoic acid enhances ferric nitrilotriacetate-induced oxidative damage in mice but not when additional alpha-tocopherol is supplemented.

Weaning mice were fed a diet supplemented with beef tallow (BT) or BT plus docosahexaenoic acid (DHA) containing 100 mg alpha-tocopherol/kg (alpha-Toc100) or 500 mg alpha-tocopherol/kg (alpha-Toc500) for 4 wk to modify membrane fatty acid unsaturation, and then were administered ferric nitrilotriacetate (Fe-NTA). The mortality caused by Fe-NTA was higher in the group fed the DHA (alpha-Toc100) diet than in the BT diet groups but the DHA (alpha-Toc500) diet suppressed this increase. Serum and kidney alpha-tocopherol contents were slightly influenced by the dietary fatty acids but not significantly. These results indicate that the increased unsaturation of tissue lipids enhances oxidative damage induced by Fe-NTA in mice fed DHA (alpha-Toc100) but not when additional alpha-tocopherol is supplemented. The apparent discrepancy between the observed enhancement by dietary DHA of oxidative damage and the beneficial effects of dietary DHA on the so-called free radical diseases is discussed in terms of strong bolus oxidative stress and moderate chronic oxidative stress.