Toxicity of oxidized fish oil in pregnancy: a dose-response study in rats.

Fish oil (FO) supplements are consumed during pregnancy to increase dietary omega-3. However, FO is often oxidized past recommended limits. In rats, a large dose of highly oxidized FO substantially increased newborn mortality, but the effects of human-relevant doses of less oxidized oil are unknown. A dose-response study in rats was conducted to estimate the safe level of oxidation during pregnancy. Sprague-Dawley rat dams were mated, then individually housed and provided with a gel treatment on each day of pregnancy. Treatment groups differed only in the FO content of the gel; control (no oil), PV5, PV10, and PV40 [0.05 mL of FO oxidized to a peroxide value (PV) of 5, 10, or 40 meq/kg], or PV40(1 mL) (1 mL of PV40). A subset of dams was culled on gestational day 20 to enable sampling, and the remainder were allowed to give birth. Newborn mortality was recorded. Offspring were sampled on postnatal days 2 and 21, and dams on day 21. There were no signs of unwellness during pregnancy. However, there was markedly increased neonatal mortality affecting the PV40(1 mL) (12.8%) and PV40 (6.3%) groups, but not the control, PV5, or PV10 groups (1%-1.4%). Dietary-oxidized FO altered the expression of placental genes involved in antioxidant pathways and the production of free radicals. Highly oxidized FO was toxic in rat pregnancy leading to a marked increase in mortality even at a human-relevant dose. We observed no toxic effects of FOs with PV ≤10 meq/kg, suggesting that this is an appropriate maximum limit.

Cross-generational trans fat intake modifies BDNF mRNA in the hippocampus: Impact on memory loss in a mania animal model.

Recently, we have described the influence of dietary fatty acids (FA) on mania-like behavior of first generation animals. Here, two sequential generations of female rats were supplemented with soybean oil (SO, rich in n-6 FA, control group), fish oil (FO, rich in n-3 FA) and hydrogenated vegetable fat (HVF, rich in trans FA) from pregnancy and during lactation. In adulthood, half of each group was exposed to an amphetamine (AMPH)-induced mania animal model for behavioral, biochemical and molecular assessments. FO supplementation was associated with lower reactive species (RS) generation and protein carbonyl (PC) levels and increased dopamine transporter (DAT) levels, while HVF increased RS and PC levels, thus decreasing catalase (CAT) activity and DAT levels in hippocampus after AMPH treatment. AMPH impaired short- (1 h) and long- (24 h) term memory in the HVF group. AMPH exposure was able to reduce hippocampal BDNF- mRNA expression, which was increased in FO. While HVF was related to higher trans FA (TFA) incorporation in hippocampus, FO was associated with increased percentage of n-3 polyunsaturated FA (PUFA) together with lower n-6/n-3 PUFA ratio. Interestingly, our data showed a positive correlation between brain-derived neurotrophic factor (BDNF) mRNA and short- and long-term memory (r(2) = 0.53; P = 0.000/r(2) = 0.32; P = 0.011, respectively), as well as a negative correlation between PC and DAT levels (r(2) = 0.23; P = 0.015). Our findings confirm that provision of n-3 or TFA during development over two generations is able to change the neuronal membrane lipid composition, protecting or impairing the hippocampus, respectively, thus affecting neurothrophic factor expression such as BDNF mRNA. In this context, chronic consumption of trans fats over two generations can facilitate the development of mania-like behavior, so leading to memory impairment and emotionality, which are related to neuropsychiatric conditions.

Toxicological evaluation of a fish oil concentrate containing Very Long Chain Fatty Acids.

Very long chain fatty acids (VLCFA) have a chain length ≥24 carbons. Fish contain low levels of these fatty acids. A commercial oil called EPAX® Evolve 05 with an up-concentration of VLCFAs of approximately 10 times, has been developed as a dietary supplement by Epax Norway AS. A series of toxicological studies were performed using mice and rats to determine the safety and toxicity of repeat dosing with a gavage administered VLCFA formulation. The results suggest transient lipid accumulation in kidneys and liver. Lipid accumulation was seen with the test item and with the soya control but was not dose related. Liver and kidney lipid accumulation, whilst present in 14- day repeat dose study, was absent in a 90-day rat study. No treatment-effect was seen in urine analysis in any of the studies. No treatment-related effects were seen with a functional observation battery, ophthalmological examination, haematology, urine analysis, oestrus cycle, thyroid hormones, organ weight, or histopathology. In the 90-day study the liver enzymes ALP, AST and ALT were statistically significantly increased with test item but within control values. There were no associated histological findings in the liver suggesting there was no toxic effect and the normalisation of values for all liver enzymes in the recovery groups suggests an adaptive response rather than a prevailing toxic response. The no-observed-adverse-effect level (NOAEL) was determined as 1200 mg VLCFA/kg b.w./day.

Safety assessment of fish oil green extraction and in vivo acute toxicity evaluation.

Sardine co-products can represent an interesting source of bioactive compounds, such as polyunsaturated fatty acids and in particular omega-3. This study aimed to investigate extraction of oil from sardine co-products by enzymatic hydrolysis using two proteases: commercial Alcalase and protease Bb from a local fungal strain (P2) of Beauveria bassiana, which overproduces proteases. Despite a higher degree of hydrolysis (41.34%) than Alcalase (24.28%), protease Bb allowed the extraction of approximately the same oil content. Resulting oil from both processes had the same fatty acid profile. Interestingly, the all-produced oil displayed an attractive w6/w3 ratio, an indicator of nutritional quality, of the order of 0.16. The safety of the generated oils was also assessed by treating two groups of Wistar rats with the fish oil administered by oral gavage at the doses (30 mg/kg and 300 mg/kg body weight) for 14 days using olive oil as a vehicle. Compared to controls used, both treated groups showed no statistically significant differences. Consequently, the acute oral toxicity evaluated by hematological, biochemical, and histological studies showed the safety of the oil generated using B. bassiana protease.

Safety assessment of EPA-rich triglyceride oil produced from yeast: genotoxicity and 28-day oral toxicity in rats.

The 28-day repeat-dose oral and genetic toxicity of eicosapentaenoic acid triglyceride oil (EPA oil) produced from genetically modified Yarrowia lipolytica yeast were assessed. Groups of rats received 0 (olive oil), 940, 1880, or 2820 mg EPA oil/kg/day, or fish oil (sardine/anchovy source) by oral gavage. Lower total serum cholesterol was seen in all EPA and fish oil groups. Liver weights were increased in the medium and high-dose EPA (male only), and fish oil groups but were considered non-adverse physiologically adaptive responses. Increased thyroid follicular cell hypertrophy was observed in male high-dose EPA and fish oil groups, and was considered to be an adaptive response to high levels of polyunsaturated fatty acids. No adverse test substance-related effects were observed on body weight, nutritional, or other clinical or anatomic pathology parameters. The oil was not mutagenic in the in vitro Ames or mouse lymphoma assay, and was not clastogenic in the in vivo mouse micronucleus test. In conclusion, exposure for 28 days to EPA oil derived from yeast did not produce adverse effects at doses up to 2820 mg/kg/day and was not genotoxic. The safety profile of the EPA oil in these tests was comparable to a commercial fish oil.

Safety assessment of EPA-rich oil produced from yeast: Results of a 90-day subchronic toxicity study.

The safety of eicosapentaenoic acid (EPA) oil produced from genetically modified Yarrowia lipolytica yeast was evaluated following 90 days of exposure. Groups of rats received 0 (olive oil), 98, 488, or 976 mg EPA/kg/day, or GRAS fish oil or deionized water by oral gavage. Rats were evaluated for in-life, neurobehavioral, anatomic and clinical pathology parameters. Lower serum cholesterol (total and non-HDL) was observed in Medium and High EPA and fish oil groups. Lower HDL was observed in High EPA and fish oil males, only at early time points. Liver weights were increased in High EPA and Medium EPA (female only) groups with no associated clinical or microscopic pathology findings. Nasal lesions, attributed to oil in the nasal cavity, were observed in High and Medium EPA and fish oil groups. No other effects were attributed to test oil exposure. Exposure to EPA oil for 90 days produced no effects at 98 mg EPA/kg/day and no adverse effects at doses up to 976 mg EPA/kg/day. The safety profile of EPA oil was comparable to that of GRAS fish oil. These results support the use of EPA oil produced from yeast as a safe source for use in dietary supplements.

Dietary n-3 polyunsaturated fatty acids enhance metastatic dissemination of murine T lymphoma cells.

Epidemiological investigation and animal studies have shown that dietary n-3 PUFA prevent the development and progression of certain types of cancer. However, conflicting results have been reported by the few studies that focused on the effect of dietary n-3 PUFA on the development of metastases. In the present study, we investigated the metastatic dissemination of murine T lymphoma lines with different metastatic potential transplanted into mice fed a fish oil diet, compared with mice fed a maize oil diet. Transplantation of highly metastatic S11 cells into animals fed a fish oil diet induced a large lymphomatoid infiltration in the spleen, associated with an eight-fold increase in spleen weight, compared with normal animals on the same diet. In contrast, only a limited increase in spleen weight was found in animals transplanted with S11 cells while fed a maize oil diet. No significant increase in spleen weight was found in animals transplanted with low-metastatic 164T2 cells regardless of whether they were fed a fish oil or a maize oil diet. At the end of experiment, an overt cachexia was shown by animals fed a fish oil diet transplanted with S11 cells, but not by those transplanted with 164T2 cells. The particularly high pro-metastatic effect of dietary n-3 PUFA on S11 cells rules out the generalisation that dietary n-3 PUFA inhibit tumour growth and progression.

Differential induction of cytochrome P450 1A1 and 1B1 mRNA in primary cultured bovine hepatocytes treated with TCDD, PBDD/Fs and feed ingredients.

This study investigates the dose-dependent expression of CYP1A1 and CYP1B1 in primary cultured bovine hepatocytes exposed to TCDD, several polybrominated dibenzo-p-dioxins and furans (PBDD/Fs) congeners and fish oil used as animal feed ingredients to identify their dioxin-like potentials. Hepatocytes were isolated from calf liver, cultured and treated for 24h with the target compounds or extracts. Quantitative real time polymerase chain reaction analysis (qRT-PCR) showed that relative mRNA levels for CYP1A1 and CYP1B1 exhibited a dose-dependent induction by TCDD. The EC(50) of the TCDD concentration for CYP1A1 expression was approximately 4-fold less than that of CYP1B1. The estimated dioxin-like toxic potential of PBDD/Fs could be ranked in the following order: 2,3,7,8-TBDD>1,2,3,7,8-PBDF>2,3,4,7,8-PBDF>1,2,3,6,7,8-HBDD. A good correlation was also observed in HRGC/HRMS-derived TEQs in fish oil samples and relative CYP1A1 mRNA induction in bovine hepatocytes treated with purified fish oil extracts. The data suggested that quantification of biomarker regulations in primary cultured hepatocytes could represent an effective tool for both the screening and study of various chemical entities in larger animals.

Negative confounding in the evaluation of toxicity: the case of methylmercury in fish and seafood.

In observational studies, the presence of confounding [corrected] can distort the true association between an exposure and a toxic-effect outcome, if the confounding variable is not controlled for in the study design or analysis phase. While confounding is often assumed to occur in the same direction as the toxicant exposure, the relationship between the benefits and risks associated with fish and seafood consumption is a classic example of negative confounding: the exposure to methylmercury occurs with fish and seafood, which are also associated with beneficial nutrients, and the signs of mercury toxicity [corrected] Mercury and nutrients may affect the same epidemiological outcomes, but most studies addressing one of them have ignored the potential for negative confounding by the other. This article reviews the existing evidence of effects of both nutrient and contaminant intakes as predictors of neurodevelopmental and cardiovascular outcomes. Substantial underestimation of the effects of mercury toxicity and of fish benefits occurs from the lack of confounder adjustment and imprecision of the exposure parameters. Given this inherent bias in observational studies, regulatory agencies should reconsider current dietary advice in order to provide better guidance to consumers in making prudent choices to maintain a nutritious diet with seafood that is low in mercury concentrations. Attention should also be paid to the occurrence of negative confounding in other connections.

Effects of three biodiesels and a low sulfur diesel in male rats--a pilot 4-week oral study.

Because of the accessible and renewable nature of feedstock and the potential for the reduction of harmful combustion emissions and greenhouse gases, biodiesels have received increasing interest as an alternate fuel. Oral exposure to biodiesels is a concern because of contact during refuelling, accidental ingestion and exposure through ground water contamination. Although biodiesels from various feedstock are in use commercially and experimentally, very little is known about their potential adverse effects and no data is available on their potential for ground water contamination. A study was performed on male rats following oral treatment with experimental biodiesels (dissolved in corn oil) derived from canola oil (Bio-C), soy oil (Bio-S) and fish oil (Bio-F), at 500 mg/kg body weight/day, 5 days per week, for 4 weeks. Separate groups of animals were treated with low sulfur diesel (LSD) for comparison purpose, and with corn oil alone to serve as control. The potential for ground water contamination by biodiesels was investigated by the preparation of water-accommodated fractions (WAF) followed by gas chromatographic analysis. WAF from Bio-F and Bio-S was found to have the highest level of dichloromethane extractable materials. Gas chromatographic analysis indicated that the extractable materials from biodiesels contained much higher proportion of C15-C30 materials than LSD. Increased liver weight was observed in animal treated with Bio-C, Bio-S and LSD and decreased thymus weight was found in those treated with Bio-S. Histopathological changes typical of male-rat specific hyaline-droplet nephropathy were detected in kidney tubules of animals treated with LSD, Bio-S and Bio-C. Mild adaptive changes were observed in thyroids of animals treated with LSD, Bio-S and Bio-F. Clinical chemical and biochemical changes were confined to Bio-S and LSD treated rats and included elevation in some hepatic phase-I and phase-II drug metabolizing enzymes and hepatic palmitoyl Co-A oxidase, and elevated urinary concentrations of ascorbic acid and albumin. At the given dose level of 500 mg/kg bw/day, the overall treatment-related effects of biodiesels and LSD are mild, and the severity of the treatment effects may be ranked as: LSD>Bio-S>Bio-C>Bio-F. Considered together with the presence of a higher level of water extractable materials, Bio-S may be more of a concern for potential human health than Bio-C and Bio-F in an oral exposure scenario. Further studies are needed to identify and characterize the constituents contributing to the treatment-related effects specific to these experimental biodiesels.

Acute toxicity of the water-soluble fraction of spent lubricating oil on the African catfish Clarias gariepinus.

Static tests were employed to assess the acute toxicity of the water-soluble fraction (WSF) of spent automotive lubricating oil (of mixed SAE grades) on Clarias gariepinus, a freshwater fish commonly cultured in Nigeria. Median lethal concentrations (LC50) of the WSF were found to decrease as a function of exposure time from 690+/-21 (after 24 h) to 513+/-58 mg/l (after 96 h). The characteristics of the WSF such as mean acidity (pH 6.6), turbidity (40 NTU), total dissolved solids (TDS; 40 mg/l) and significantly reduced (P<0.05) dissolved-oxygen (DO) values (1.44 mg/l) were not compliant with existing standards set for discharged effluents. The solubility of the detected straight-chain aliphatics ranked as C14>C16>C32>C18>C28; that of the simple aromatics was ortho-xylene>para-xylene; and that of the polyaromatic hydrocarbons (PAHs) was acenaphthylene>9H-fluorene>naphthalene>anthracene>phenanthrene>chrysene>benzo[k]fluoranthene>benzo[a]pyrene>benzo[b]fluoranthene, most of which being serious carcinogens. These oil constituents and the overall physico-chemical properties of the WSF are expected to act synergistically on the test organism (C. gariepinus), eliciting the quantal responses observed. The toxicity of the WSF points to the base constituent, oxidative degradation, and mechano-chemical reactions associated with aged crankcase oils. These oils, therefore, should definitely no longer be disposed into water streams or landscape, not even at sub-lethal concentrations, because of the inherent toxicity of their soluble fractions and the associated danger of bioaccumulation.

Murine strain differences in 8-hydroxy-deoxyguanosine formation in hepatic DNA induced by oxidized lard and dietary oils.

Difference of 8-hydroxy-deoxyguanosine (8-OH-dG) formation in liver DNA in C3H/HeN and in C57BL/6 mice--fed oxidized lard and dietary oils (soybean and sardine)--was investigated. The blank levels of 8-OH-dG were higher in C3H/HeN mice (highly sensitive to liver tumorigenesis) than in C57BL/6 mice (resistant strain). The level of 8-OH-dG increased much more in C3H/HeN mice than in the C57BL/6 mice fed by oxidized lard and dietary oil treatment. Feeding oxidized lard and dietary oils increased 8-oxo-guanine DNA glycosylase I (OGG1) and mRNA 8-oxo-dGTPase in C57BL/6 mice. On the other hand, no appreciable change of mRNA in the C3H/HeN mice was observed. The formation differences of 8-OH-dG from the two murine strains fed with oxidized lard and dietary oils may be associated with the different mRNA levels in the DNA repair enzymes because the mRNA levels in the DNA repair enzymes were much lower in C3H/HeN mice than in C57BL/6 mice.

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.

Skipjack tuna (Katsuwonus pelamis) eyeball oil exerts an anti-inflammatory effect by inhibiting NF-κB and MAPK activation in LPS-induced RAW 264.7 cells and croton oil-treated mice.

The effect of tuna eyeball oil (TEO) on lipopolysaccharide (LPS)-induced inflammation in macrophage cells was investigated. TEO had no cytotoxicity in cell viability as compared to the control in LPS induced RAW 264.7 cells. TEO reduced the levels of NO and pro-inflammatory cytokines by up to 50% in a dose-dependent manner. The expression of NF-κB and MAPKs as well as iNOS and COX-2 proteins was reduced by TEO, which suggests that its anti-inflammatory activity is related to the suppression of the NF-κB and MAPK signaling pathways. The rate of formation of ear edema was reduced compared to that in the control at the highest dose tested. In an acute toxicity test, no mice were killed by TEO doses of up to 5000mg/kg body weight during the two week observation period. These results suggested that TEO may have a significant effect on inflammatory factors and be a potential anti-inflammatory therapeutic.

Subchronic toxicity of baltic herring oil and its fractions in the rat (III) bone tissue composition and dimension, and ratio of n-6/n-3 fatty acids in serum phospholipids.

Changes in total bone mineral density determined by the bone-ash method were recently demonstrated in rats, exposed to Herring oil from the contaminated southern part of the Baltic Sea. In the present study more detailed analysis of bone structure and biomechanics was performed and obtained results were evaluated in the context of dietary factors, such as polyunsaturated fatty acids, vitamin D and vitamin A. Baltic Sea herring oil was fractionated into one relatively pollutant-free fraction (F1), and two fractions with pronounced enrichment of pollutants (F2 and F3). Female Sprague-Dawley rats were fed diets supplemented with Baltic Herring oil, its fractions, Nordic Sea capelin oil or soy oil. Femur was scanned with peripheral quantitative computed tomography (pQCT) and also tested by a mechanical compression analysis. Polyunsaturated fatty acids, vitamin A and D were analysed in serum. Rats fed the high dose of herring oil exhibited shorter femur length with decreased diaphyseal cortical bone mineral density, as well as lowered metaphyseal cross-sectional area compared to the soy oil group. Rats fed the high dose of F1 diet had increased cortical and decreased trabecular area, and higher total and trabecular bone mineral density. Rats fed the low dose of F2 diet showed similar changes associated with increased maximum load and energy absorption in compression test of the femoral metaphysis. In summary, our findings in changes of bone geometry and density could not be linked to any isolated exposure parameter, suggesting synergistic or antagonistic effects of several components of the test diets.

Beneficial effect of an omega-6 PUFA-rich diet in non-steroidal anti-inflammatory drug-induced mucosal damage in the murine small intestine.

AIM: To investigate the effect of a fat rich diet on non-steroidal anti-inflammatory drug (NSAID)-induced mucosal damage in the murine small intestine. METHODS: C57BL6 mice were fed 4 types of diets with or without indomethacin. One group was fed standard laboratory chow. The other groups were fed a fat diet consisting of 8% w/w fat, beef tallow (rich in SFA), fish oil, (rich in omega-3 PUFA), or safflower oil (rich in omega-6 PUFA). Indomethacin (3 mg/kg) was injected intraperitoneally from day 8 to day 10. On day 11, intestines and adhesions to submucosal microvessels were examined. RESULTS: In the indomethacin-treated groups, mucosal damage was exacerbated by diets containing beef tallow and fish oil, and was accompanied by leukocyte infiltration (P < 0.05). The mucosal damage induced by indomethacin was significantly lower in mice fed the safflower oil diet than in mice fed the beef tallow or fish oil diet (P < 0.05). Indomethacin increased monocyte and platelet migration to the intestinal mucosa, whereas safflower oil significantly decreased monocyte and platelet recruitment (P < 0.05). CONCLUSION: A diet rich in SFA and omega-3 PUFA exacerbated NSAID-induced small intestinal damage via increased leukocyte infiltration. Importantly, a diet rich in omega-6-PUFA did not aggravate inflammation as monocyte migration was blocked.

Fish oil feeding results in an enhancement of cholesterol-induced atherosclerosis in rabbits.

We investigated the influence of fish oil on cholesterol induced atherosclerosis in rabbits. Group I, a control group was fed a cholesterol-free diet, group II was fed a diet supplemented with 1.5% cholesterol, group III received in addition to cholesterol supplementation a purified fish oil concentrate (Maxepa, 2 ml/d). The animals received these diets for 5 months (100 g/d). Aortic atherosclerosis as measured by planimetry of sudanophilic lesions was significantly higher (+59%) in group III as compared with group II, even though serum cholesterol levels were comparable. No differences were found in platelet half-life times between groups II and III, but these values were significantly lower than the half-life of platelets in the control group I. Total serum peroxide levels, expressed as malondialdehyde equivalents were significantly elevated in the fish oil-treated group. This may be due to malondialdehyde modification of the lipoproteins and may be responsible for the enhanced development of atherosclerosis in these animals.

Effects of a fish oil rich diet on hyperoxic lung damage in mice.

Mice were fed a chow diet plus 10% cellulose, 10% fish oil or 10% sunflower oil for 3 weeks, then exposed to 100% oxygen for 75 h. Large changes in lung fatty acid composition occurred, but this did not affect hyperoxic lung damage nor levels of thiobarbituric acid reactive substances or myeloperoxidase in lungs of mice following exposure to hyperoxia. Thus there is no evidence that the ingestion of large quantities of fish oil increased the susceptibility to the oxidative stress induced by hyperoxia.

Ornithine decarboxylase and thymidine kinase activities and polyamine levels from selected organs of adult miniature swine receiving three concentrations of dietary menhaden oil.

Mature, female swine were randomly assigned to one of seven dietary groups. Swine in groups 1-3 were fed a cholesterol-rich diet for 55 days while the remaining groups remained on a basal swine diet. At the end of the cholesterol(Chol)-preloading period the swine in groups 1-7 were placed on menhaden oil (MO) and/or corn oil (CO) as follows: groups 1 and 4, 15% CO (control); groups 2 and 5, 0.75% MO+14.25% CO; groups 3 and 7, 15% MO; and group 6, 7.5% MO+7.5% CO. Animals were killed at the end of the approximately 6-month feeding period and portions of liver, pancreas and colon mucosa were analyzed for both ornithine decarboxylase (ODC) and thymidine kinase (TK) activity while polyamine levels were measured in the liver and pancreas. Statistical analyses were carried out by one-way and two-way ANOVA and by trend analysis. In the pancreas, the highest MO group (group 7) had significantly higher ODC levels when compared with the CO control (group 4) and the next to highest MO group (group 6) (one-way ANOVA)-all non-cholesterol preloaded groups. Using a two-way ANOVA (Chol-by-MO), liver ODC was significantly lower in the CO control when compared with the lowest and highest MO groups (groups 5 and 7, respectively), again in the non-cholesterol-preloaded animals. In the colon, the swine in the Chol-low MO group (group 2) had significantly lower TK activity than the Chol/CO control group (group 1) and Chol/Hi MO group (group 3) (one-way ANOVA) and also had significantly lower activity than all groups except the CO control (group 4) (two-way ANOVA). Liver acetylputrescine in the lowest and highest MO groups (groups 5 and 7, respectively) was significantly higher than in the CO group (group 4). Liver spermidine in the Chol-Hi MO group (group 3) was significantly higher than the Chol-Lo MO group (group 2), while the highest MO group (group 7) had a statistically higher level than the other non-cholesterol groups (groups 4-6) (one-way ANOVA). Liver spermine was significantly higher in the Chol-Hi MO group (group 3) when compared to the CO control (group 1) and the Chol-Lo MO group (group 2) (one-way ANOVA). Pancreatic putrescine in the CO control (group 4) was significantly higher than all other groups (two-way ANOVA) while spermine from the 2 Chol-MO groups (groups 2 and 3) was higher than the Chol-CO control (group 1) (one-way ANOVA). Using trend analysis, liver TK, putrescine and spermidine increased in the non-cholesterol preloaded groups with increasing dietary MO, similar to the increase seen in ODC. Thus, of the three organs studied, only liver responded to menhaden oil with changes in both ODC itself or some of its metabolic engendered products and thymidine kinase; at least for one of the parameters, ODC, change associated with dietary MO was dependent on whether the swine were preloaded with cholesterol.

Preliminary safety assessment of an arachidonic acid-enriched oil derived from Mortierella alpina: summary of toxicological data.

An arachidonic acid-enriched oil (AA-oil), derived from Mortierella alpina was subjected to a programme of studies to establish its preliminary safety for use in infant nutrition. This was addressed at two levels: (1) HPLC analysis of metabolites produced by the production strains at various conditions, and (2) an evaluation of the toxicity of the final product. The following studies were carried out on the AA-oil: gene mutation assays in bacteria and mammalian cells in vitro; chromosome aberration assays both in vitro and in vivo and acute and subacute (4-wk) oral toxicity in the rat. No known mycotoxins were produced by the production strains under the conditions tested. Further, the oil did not show mutagenic or clastogenic activity and the acute oral toxicity, expressed as the LD50 value, exceeded 20 ml/kg body weight, that is, 18.2 g/kg body weight. In the subacute oral toxicity study the AA-oil was tested as such and in combination with a docosahexaenoic-enriched oil (DHA-oil) derived from fish oil at a ratio of 2:1 (AA:DHA). This was done because high dose levels of AA may result in adverse effects; DHA can compensate for these effects. Furthermore, human milk contains both AA and DHA at a ratio of AA:DHA of 2 to 3:1. No obvious signs of toxicity were observed. Levels of phospholipids and triglycerides tended to be decreased in the highest dose groups. The no-observed-adverse-effect level of the AA-oil in the subacute 4-wk toxicity study was placed at the highest levels tested, namely 3000 mg AA-oil/kg body weight/day as such and in the combination of 3000 mg AA-oil and 1500 mg DHA-oil/kg body weight/day. This corresponds to an intake of 1000 mg AA/kg body weight/day, which represents approximately 37 times the infant intake of AA in human milk.