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.

In vitro genotoxicity assessment of functional ingredients: DHA, rutin and α-tocopherol.

The in vitro genotoxicity of three compounds widely used as functional ingredients, docosahexaenoic acid (DHA), rutin and α-tocopherol, was assessed. A miniaturized version of the Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of OECD 471), and the in vitro micronucleus test in TK6 cells (OECD 487) were performed. This strategy is recommended by the European Food Safety Authority for the in vitro genotoxicity assessment of food and feed. In addition, this approach was complemented with the in vitro standard and enzyme-modified comet assay (S9-/S9+) using hOGG1, EndoIII and hAAG in order to assess potential premutagenic lesions in TK6 cells. Rutin showed an equivocal response in the in vitro micronucleus test and also was a potent Salmonella typhimurium revertant inductor in the Ames test. DHA showed equivocal results in the in vitro micronucleus test. In this regard, DHA and rutin seemed to interact with the DNA at a chromosomal level, but rutin is also capable of producing frameshift mutations. No genotoxicity was observed in cells treated with α-tocopherol. This article complements the evidence already available about the genotoxicity of these compounds. However, more studies are needed in order to elucidate the consequences of their use as functional ingredients in human health.

Embryo-toxicity of docosahexaenoic and eicosapentaenoic acids: In vivo and in silico investigations using the chick embryo model.

OBJECTIVE: The objective of the current study was to evaluate the embryo-toxicity of omega-3 fatty acids. METHODS: Firstly, the embryo-toxicity of docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), as well as their interaction with Bcl-2 family members, were predicted using an in silico assay. In the next step, the embryonic pathological lesions and amniotic fluid biochemical changes following omega-3 treatment were investigated using a chick embryo model. Finally, the drug's vascular apoptotic effect on the chick's yolk sac membrane (YSM) was assessed. RESULTS: In silico simulations revealed the embryo-toxicity, tissue-toxicity (respiratory and cardiovascular), and vascular-toxicity (apoptotic activity) of DHA and EPA. There was also an accurate interaction between DHA and EPA with Bax (Binding affinity: -7.6 and -10.6 kcal/mol) and Bcl-2 (Binding affinity: -8.0 and -12.2 kcal/mol), respectively. Moreover, DHA and EPA administrations were related to various adverse consequences, including weight loss and lesions in the respiratory and cardiovascular systems. Histopathological findings consisted of pulmonary edema, airway dilatation, increased interstitial tissue, and hyperemia in the lungs, heart, liver, kidney, and brain. Morphometric evaluation of the YSM vasculature revealed that the vascular apoptotic effect of omega-3was associated with a significant reduction in mean capillary area. In immunohistochemistry assay, increased expression of BAX and low expression of Bcl-2 affirmed apoptosis in YSM vessels. CONCLUSION: According to the results of this study, one could confirm that the possible embryo-toxicity of omega-3 was approved by data presented in this research. The obtained results also support the suspicion that alteration of the apoptotic-related proteins in vessels is an essential pathway in embryo-toxicity of omega-3.

Toxicological evaluation of an unextracted Aurantiochytrium limacinum biomass, a novel docosahexaenoic acid rich feed ingredient.

The purpose of this paper was to evaluate the toxicological potential of a heterotrophically grown unextracted Aurantiochytrium limacinum biomass (AURA) when used as a food additive. The following toxicological assessments were conducted on this novel docosahexaenoic acid rich feed ingredient: Mutagenic activity was tested by means of the Ames' test using five Salmonella typhimurium strains; clastogenic activity was investigated using the micronucleus test in male and female Sprague Dawley rats; genotoxic activity was assessed by means of the in vitro metaphase analysis tests in human lymphocytes; oral toxicity was tested by administration of AURA at various concentrations; eye and skin irritation was assessed in rabbits according to OECD guidelines; skin sensitivity was established in guinea pigs by means of the Buehler test. All conducted tests were considered valid under the experimental conditions. No significant mutagenic activity or clastogenic activity was observed. Genotoxic activity in human lymphocytes was not induced. Oral administration of 276 mg AURA/kg bw1 and 2000 mg AURA/kg bw resulted in no mortality or signs of acute toxicity. Daily administration of 1000 mg AURA/kg bw caused no mortality or biologically relevant signs of toxicity and was established as the No Observable Adverse Effect Level. AURA was also found to be a non-irritant for the eye and skin of the rabbit and was non-sensitizing to guinea pig skin.

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.

Non-enzymatic conversion of primary oxidation products of Docosahexaenoic acid into less toxic acid molecules.

Docosahexaenoic acid (DHA) is long chain omega-3 fatty acid with known health benefits and clinical significance. However, 4-hydroxy hexenal (HHE), an enzymatic oxidation product of DHA has recently been reported to have health-damaging effects. This conflict raises major concern on the long-term clinical use of these fatty acids. Even though the enzymatic and non-enzymatic conversion of HHE to nontoxic acid molecules is possible by the aldehyde detoxification systems, it has not yet studied. To address this, primary oxidation products of DHA in lipoxidase system were subjected to non-enzymatic conversion at physiological temperature over a period of 1 week. The reaction was monitored using HPLC, IR spectroscopy and biochemical assays (based on the loss of conjugated dienes, lipid peroxides aldehydes). Short term and long term cytotoxicity of the compounds generated at various time points were analyzed. IR and HPLC spectra revealed that the level of aldehydes in the primary oxidation products reduced over time, generating acids and acid derivatives within a week period. In short term and long term cytotoxicity analysis, initial decomposition products were found more toxic than the 1-week decomposition products. Further, when primary oxidation products were subjected to aldehyde dehydrogenase mediated oxidation, it generated products that are also less toxic. The study suggests the possible non-enzymatic conversion of primary oxidation products of DHA to less cytotoxic acid molecules. Exploration of the physiological roles of these acid molecules may explain the biological potential of omega-3 fatty acids.

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.

Developmental and reproductive toxicological evaluation of arachidonic acid (ARA)-Rich oil and docosahexaenoic acid (DHA)-Rich oil.

The purpose of this study was to investigate the reproductive and developmental toxicity of dietary exposure to DHA-rich oil from Schizochytrium sp. and ARA-rich oil from Mortierella alpina. In a developmental toxicity study, pregnant Wistar rats were untreated (control) or administered corn oil (vehicle control), 1000, 2500, or 5000 mg/kg bw/day of DHA-rich oil or ARA-rich oil via gavage from gestation days 6 through 20. In the reproductive toxicity study, male and female Wistar rats were administered vehicle control (corn oil), or 1000, 2500, or 5000 mg/kg bw/day of DHA- or ARA-rich oil via gavage throughout the mating period, pregnancy, and the nursing and lactation period. Differences in the number of fetuses, fetal skeletal malformations, and external and visceral anomalies in the developmental study and mortality, clinical signs, fertility indices, physical observations, gross necropsy findings, and gestation period length in the reproductive toxicity study were not dose-related or significantly different from control groups, and were not considered to be treatment related. The no observed adverse effect level (NOAEL) for maternal toxicity and embryo/fetal development and for paternal or maternal treatment-related reproductive toxicity for the DHA-rich oil and ARA-rich oil administered by gavage, was 5000 mg/kg bw/day.

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.

Investigation into the distinct subcellular effects of docosahexaenoic acid loaded low-density lipoprotein nanoparticles in normal and malignant murine liver cells.

BACKGROUND: Recent studies have shown that low density lipoproteins reconstituted with the natural omega 3 fatty acid docosahexaenoic acid (LDL-DHA) is selectively cytotoxic to liver cancer cells over normal hepatocytes. To date, little is known about the subcellular events which transpire following LDL-DHA treatment. METHODS: Herein, murine noncancer and cancer liver cells, TIB-73 and TIB-75 respectively, were investigated utilizing confocal microscopy, flow cytometry and viability assays to demonstrate differential actions of LDL-DHA nanoparticles in normal versus malignant cells. RESULTS: Our studies first showed that basal levels of oxidative stress are significantly higher in the malignant TIB-75 cells compared to the normal TIB-73 cells. As such, upon entry of LDL-DHA into the malignant TIB-75 cells, DHA is rapidly oxidized precipitating global and lysosomal lipid peroxidation along with increased lysosomal permeability. This leakage of lysosomal contents and lipid peroxidation products trigger subsequent mitochondrial dysfunction and nuclear injury. The cascade of LDL-DHA mediated lipid peroxidation and organelle damage was partially reversed by the administration of the antioxidant, N-acetylcysteine, or the iron-chelator, deferoxamine. LDL-DHA treatment in the normal TIB-73 cells was well tolerated and did not elicit any cell or organelle injury. CONCLUSION: These studies have shown that LDL-DHA is selectively cytotoxic to liver cancer cells and that increased levels of ROS and iron catalyzed reactions promote the peroxidation of DHA which lead to organelle dysfunction and ultimately the demise of the cancer cell. GENERAL SIGNIFICANCE: LDL-DHA selectively disrupts lysosomal, mitochondrial and nuclear function in cancer cells as a novel pathway for eliminating cancer cells.

Toxicological evaluation of arachidonic acid (ARA)-rich oil and docosahexaenoic acid (DHA)-rich oil.

The safety of DHA-rich oil from Schizochytrium sp. and ARA-rich oil from Mortierella alpina was separately evaluated by testing for gene mutations, clastogenicity, and aneugenicity, and by conducting 28-day and 90-day dietary studies in Wistar rats. The results of all genotoxicity tests were negative. The 28-day and 90-day studies involved dietary exposure to 1000, 2500, and 5000 mg per kg bw of the DHA-rich and ARA-rich oils and two control diets: water and corn oil (vehicle control). There were no treatment-related effects of either the DHA-rich or ARA-rich oils on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, coagulation, urinalysis parameters, or necropsy findings. Increases in cholesterol and triglyceride levels were considered related to a high oil diet and non-adverse. The no observable adverse effect level (NOAEL) for both the DHA-rich and ARA-rich oils was 5000 mg per kg bw, the highest dose tested. The results confirm that these oils possess toxicity profiles similar to those of other currently marketed oils and support the safety of DHA-rich oil from Schizochytrium sp. and ARA-rich oil from Mortierella alpina for their proposed uses in food.

Enhanced anti-inflammatory effects of DHA and quercetin in lipopolysaccharide-induced RAW264.7 macrophages by inhibiting NF-κB and MAPK activation.

The aim of the present study was to investigate the anti-inflammatory effects of docosahexaenoic acid (DHA) + quercetin (QE) used in combination. DHA and QE are natural compounds derived from various foods and have been demonstrated to exert anti­inflammatory effects The protein mRNA expression involved in the nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signalling pathway was analyzed by western blot analysis and reverse transcription-polymerase chain reaction methods respectively, other cytokines were detected by an enzyme­linked immunosorbent assay kit. The results of the present study demonstrated that combined treatment of lipopolysaccharide (LPS)­stimulated RAW264.7 cells with DHA + QE decreased the levels of pro­inflammatory mediators to a greater extent than QE or DHA alone. Additionally, DHA + QE synergistically suppressed nitric oxide, prostaglandin E2 and cyclooxygenase-2 levels. Molecular­level studies indicated that the DHA + QE combination can significantly inhibit the mRNA expression of NF­κB subunits p50 and p65, extracellular signal­regulated kinase (ERK) 1/2 and c­JUN N­terminal kinase (JNK) 1/2, which suggests that the NF­κB signalling pathway is involved in the synergistic effects observed. Furthermore, western blot analysis demonstrated that DHA + QE synergistically inhibit the phosphorylation of p50, p65, ERK1/2 and JNK1/2. This finding indicates that the enhanced anti­inflammatory effects of the combined compounds are achieved by suppressing NF­κB and MAPK signalling in LPS­stimulated RAW264.7 cells. The results of the present study suggest that DHA and QE in combination may be utilized as potent anti­inflammatory compounds, with potential preventative or palliative effects on obesity, atherosclerosis and cardiovascular diseases.

Nucleotide excision repair deficiency increases levels of acrolein-derived cyclic DNA adduct and sensitizes cells to apoptosis induced by docosahexaenoic acid and acrolein.

The acrolein derived cyclic 1,N(2)-propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125µM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116+ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA.

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.

Toxic effects of n-3 polyunsaturated fatty acids in human lung A549 cells.

N-3 polyunsaturated fatty acids (PUFAs), particularly eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) are crucial for the prevention of lung cancer. PUFAs may act through alteration of membrane fluidity and cell surface receptor functions; modulation of cyclooxygenase activity; and increased cellular oxidative stress, which may induce apoptosis and autophagy. Therefore the aim of the study was to investigate whether EPA and DHA (25-100 µM) are able to reduce human lung cancer cell growth through oxidative stress influence on autophagy and apoptosis. It was found that both EPA and DHA in the concentration-dependent manner suppressed the cell viability, enhanced cell death, induced activation of caspase-3/7 and potentiated intracellular oxidative DNA and protein damage. In response to PUFAs intracellular autophagic vacuolization occurred and the observed effect was reverted when the autophagy inhibitor 3-methyladenine (3-MA) was applied. The inhibition of the autophagic process enhanced the cell viability, suppressed cell death, and decreased activation of caspase-3/7 indicating that EPA and DHA-induced autophagy amplified A549 apoptotic cell death.

The HR96 activator, atrazine, reduces sensitivity of D. magna to triclosan and DHA.

HR96 is a CAR/PXR/VDR ortholog in invertebrates, and a promiscuous endo- and xenobiotic nuclear receptor involved in acclimation to toxicants. Daphnia HR96 is activated by chemicals such as atrazine and linoleic acid (LA) (n-6 fatty acid), and inhibited by triclosan and docosahexaenoic acid (DHA) (n-3 fatty acid). We hypothesized that inhibitors of HR96 may block the protective responses of HR96 based on previously performed luciferase assays. Therefore, we performed acute toxicity tests with two-chemical mixtures containing a HR96 inhibitor (DHA or triclosan) and a HR96 activator (LA or atrazine). Surprisingly, results demonstrate that triclosan and DHA are less toxic when co-treated with 20-80 µM atrazine. Atrazine provides concentration-dependent protection as lower concentrations have no effect and higher concentrations cause toxicity. LA, a weaker HR96 activator, did not provide protection from triclosan or DHA. Atrazine's protective effects are presumably due to its ability to activate HR96 or other toxicologically relevant transcription factors and induce protective enzymes. Atrazine did not significantly induce glucosyltransferase, a crucial enzyme in triclosan detoxification. However, atrazine did increase antioxidant activities, crucial pathways in triclosan's toxicity, as measured through GST activity and the TROLOX equivalence assay. The increase in antioxidant capacity is consistent with atrazine providing protection from a wide range of toxicants that induce ROS, including triclosan and unsaturated fatty acids predisposed to lipid peroxidation.

Relation of Prenatal Methylmercury Exposure from Environmental Sources to Childhood IQ.

BACKGROUND: Although prenatal methylmercury exposure has been linked to poorer intellectual function in several studies, data from two major prospective, longitudinal studies yielded contradictory results. Associations with cognitive deficits were reported in a Faroe Islands cohort, but few were found in a study in the Seychelles Islands. It has been suggested that co-exposure to another contaminant, polychlorinated biphenyls (PCBs), may be responsible for the positive findings in the former study and that co-exposure to nutrients in methylmercury-contaminated fish may have obscured and/or protected against adverse effects in the latter. OBJECTIVES: We aimed to determine the degree to which co-exposure to PCBs may account for the adverse effects of methylmercury and the degree to which co-exposure to docosahexaenoic acid (DHA) may obscure these effects in a sample of Inuit children in Arctic Québec. METHODS: IQ was estimated in 282 school-age children from whom umbilical cord blood samples had been obtained and analyzed for mercury and other environmental exposures. RESULTS: Prenatal mercury exposure was related to poorer estimated IQ after adjustment for potential confounding variables. The entry of DHA into the model significantly strengthened the association with mercury, supporting the hypothesis that beneficial effects from DHA intake can obscure adverse effects of mercury exposure. Children with cord mercury ≥ 7.5 µg/L were four times as likely to have an IQ score < 80, the clinical cut-off for borderline intellectual disability. Co-exposure to PCBs did not alter the association of mercury with IQ. CONCLUSIONS: To our knowledge, this is the first study to document an association of prenatal mercury exposure with poorer performance on a school-age assessment of IQ, a measure whose relevance for occupational success in adulthood is well established. This association was seen at levels in the range within which many U.S. children of Asian-American background are exposed.

PPARδ signaling mediates the cytotoxicity of DHA in H9c2 cells.

Docosahexaenoic acid (22:6n3, DHA) is an n-3 polyunsaturated fatty acid (PUFA) known to affect numerous biological functions. While DHA possesses many properties that impact cell survival such as suppressing cell growth and inducing apoptosis, the exact molecular and cellular mechanism(s) remain unknown. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate many cell pathways including cell death. As DHA acts as a ligand to PPARs the aim of this study was to examine the involvement of PPARδ in DHA-mediated cytotoxicity toward H9c2 cells. Treatment with DHA (100µM) resulted in a significant decline in cell viability, cellular metabolic activity and total antioxidant capacity coinciding with increased total proteasome activities and activity of released lactate dehydrogenase (LDH). No changes in reactive oxygen species (ROS) production or accumulation of lipid peroxidation products were observed but DHA promoted apoptotic cell death as detected by flow cytometry, increased caspase-3 activity and decreased phosphorylation of Akt. Importantly, DHA enhanced PPARδ DNA binding activity in H9c2 cells strongly signifying that the cytotoxic effect of DHA might be mediated via PPARδ signaling. Co-treatment with the selective PPARδ antagonist GSK 3787 (1µM) abolished the cytotoxic effects of DHA in H9c2 cells. Cytotoxic effects of DHA were attenuated by co-treatment with myriocin, a selective inhibitor of serine palmitoyl transferase (SPT), preventing de novo ceramide biosynthesis. LC/MS analysis revealed that treatment with DHA resulted in the accumulation of ceramide, which was blocked by GSK 3787. Interestingly, inhibition of cytochrome P450 (CYP) oxidase with MS-PPOH (50µM) abolished DHA-mediated cytotoxicity suggesting downstream metabolites as the active mediators. We further demonstrate that CYP oxidase metabolites of DHA, methyl epoxy docosapentaenoate (EDP methyl esters, 1µM) (mix 1:1:1:1:1:1; 4,5-, 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP methyl esters) and 19,20-EDP cause cytotoxicity via activation of PPARδ signaling leading to increased levels of intracellular ceramide. These results illustrate novel pathways for DHA-induced cytotoxicity that suggest an important role for CYP-derived metabolites, EDPs.

Pifithrin-alpha has a p53-independent cytoprotective effect on docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma HepG2 cells.

Pifithrin-alpha (PFT) is an inhibitor of p53 and is known to protect against a variety of p53-mediated genotoxic agents. In this report, we examined the inhibitory effects of PFT against docosahexaenoic acid (DHA)-induced cytotoxicity in the human hepatocellular carcinoma (HCC) cell line HepG2. PFT significantly abrogated DHA-induced cytotoxicity in wild-type HepG2 cells (normal expression of p53) and after p53-knockdown by siRNA, as well as in Hep3B (p53 null) and Huh7 (p53 mutant) cells. DHA-induced cytotoxicity is mediated by induction of oxidative stress, and PFT inhibited this event, but it does not exert antioxidant effects. PFT significantly suppressed the release of cytochrome c from mitochondria to cytosol, as well as changes in the mitochondrial membrane potential (ΔΨM) by DHA. Therefore, protection of mitochondria by PFT is crucial for its inhibition of DHA-induced cytotoxicity. Although it has been reported that PFT is able to block p53 function, our data suggest that PFT also has a p53-independent inhibition mechanism. This work provided insights into the mechanisms of PFT action on DHA-induced cytotoxicity in HCC.

Intravitreal docosahexaenoic acid in a rabbit model: preclinical safety assessment.

PURPOSE: The purpose of the present study was to evaluate the retinal toxicity of a single dose of intravitreal docosahexaenoic acid (DHA) in rabbit eyes over a short-term period. METHODS: Sixteen New Zealand albino rabbits were selected for this pre-clinical study. Six concentrations of DHA (Brudy Laboratories, Barcelona, Spain) were prepared: 10 mg/50 µl, 5 mg/50 µl, 2'5 mg/50 µl, 50 µg/50 µl, 25 µg/50 µl, and 5 µg/50 µl. Each concentration was injected intravitreally in the right eye of two rabbits. As a control, the vehicle solution was injected in one eye of four animals. Retinal safety was studied by slit-lamp examination, and electroretinography. All the rabbits were euthanized one week after the intravitreal injection of DHA and the eyeballs were processed to morphologic and morphometric histological examination by light microscopy. At the same time aqueous and vitreous humor samples were taken to quantify the concentration of omega-3 acids by gas chromatography. Statistical analysis was performed by SPSS 21.0. RESULTS: Slit-lamp examination revealed an important inflammatory reaction on the anterior chamber of the rabbits injected with the higher concentrations of DHA (10 mg/50 µl, 5 mg/50 µl, 2'5 mg/50 µ) Lower concentrations showed no inflammation. Electroretinography and histological studies showed no significant difference between control and DHA-injected groups except for the group injected with 50 µg/50 µl. CONCLUSIONS: Our results indicate that administration of intravitreal DHA is safe in the albino rabbit model up to the maximum tolerated dose of 25 µg/50 µl. Further studies should be performed in order to evaluate the effect of intravitreal injection of DHA as a treatment, alone or in combination, of different retinal diseases.