Safety evaluation of the food enzyme pectinesterase from the genetically modified Trichoderma reesei strain RF6201.

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Trichoderma reesei strain RF6201 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, production of wine and wine vinegar, coffee demucilation and production of plant extracts as flavouring preparations. Since residual amounts of the total organic solids (TOS) are removed during the coffee demucilation and the production of flavouring extracts, dietary exposure was calculated only for the remaining three food processes. It was estimated to be up to 0.532 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,880. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found with pollen allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure, particularly in individuals sensitised to pollen allergens, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme endo-polygalacturonase from the genetically modified Trichoderma reesei strain RF6197.

The food enzyme endo-polygalacturonase ((1-4)-α-d-galacturonan glycanohydrolase; EC 3.2.1.15) is produced with the genetically modified Trichoderma reesei strain RF6197 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, production of wine and wine vinegar, coffee demucilation and production of plant extracts as flavouring preparations. Since residual amounts of the total organic solids (TOS) are removed during the coffee demucilation and the production of flavouring extracts, dietary exposure was calculated only for the remaining three food processes. It was estimated to be up to 0.156 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 6,410. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and matches were found with a number of pollen allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme endo-polygalacturonase from the genetically modified Aspergillus oryzae strain AR-183.

The food enzyme endo-polygalacturonase (1→4)-α-d-galacturonan glycanohydrolase EC 3.2.1.15 is produced with the genetically modified Aspergillus oryzae strain AR-183 by AB ENZYMES GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juice, production of wine and wine vinegar, production of plant extracts as flavouring preparations and coffee demucilation. Since residual amounts of total organic solids (TOS) are removed by repeated washing or distillation, dietary exposure to the food enzyme TOS from coffee demucilation and from the production of flavouring extracts was considered not necessary. For the remaining three food processes, dietary exposure was estimated to be up to 0.087 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 11,494. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with pollen allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to pollen allergens, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme pectinesterase from the genetically modified Aspergillus oryzae strain AR-962.

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Aspergillus oryzae strain AR-962 by AB Enzymes GmbH. The genetic modifications did not give rise to safety concerns. The food enzyme was free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juice, production of wine and wine vinegar, production of plant extracts as flavouring preparations and coffee demucilation. Since residual amounts of total organic solids are removed by repeated washing or distillation, dietary exposure to the food enzyme total organic solids (TOS) from the production of flavouring extracts and coffee demucilation was considered not necessary. For the remaining three food processes, dietary exposure to the food enzyme-TOS was estimated to be up to 0.647 mg TOS/kg bw per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,546. A search for the similarity of the amino acid sequence to those of known allergens was made and two matches with pollen allergens were found. The Panel considered that, under the intended conditions of use the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen allergens, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Guidance on safety evaluation of sources of nutrients and bioavailability of nutrient from the sources (Revision 1).

[Table: see text] This guidance describes the scientific data required to allow an evaluation of the safety of new substances that are proposed for use as sources of nutrients in food supplements, foods for the general population or foods for specific groups and an assessment of the bioavailability of the nutrient from the proposed source. This guidance describes the scientific data required to allow an evaluation of the safety of the source within the established framework for risk assessment of food additives and novel food ingredients and the bioavailability of the nutrient from this source. This document is arranged in five main sections: one on technical data aimed at characterising the proposed source and at identifying potential hazards resulting from its manufacture and stability in food; one on existing authorisations and evaluation, providing an overview of previous assessments on the proposed source and their conclusions; one on proposed uses and exposure assessment section, allowing an estimate of the dietary exposure to the source and the nutrient based on the proposed uses and use levels; one on toxicological data, describing approaches which can be used to identify (in conjunction with data on manufacture and composition) and to characterise hazards of the source and any relevant breakdown products; the final section on bioavailability focuses on determining the extent to which the nutrient from the proposed source is available for use by the body in comparison with one or more forms of the same nutrient that are already permitted for use on the positive lists. This guidance was adopted by the Panel on Food Additives and Nutrient Sources added to Food (ANS Panel) on 16 May 2018. Upon request from EFSA, the present guidance has been revised to inform applicants of new provisions set out in Regulation (EC) No 178/2002, as amended by Regulation (EU) 2019/1381 on the transparency and sustainability of the EU risk assessment in the food chain.

Subacute oral toxicity investigation of selenium nanoparticles and selenite in rats.

Selenium (Se) nanoparticles have been proposed as food supplements. However, the particle formulation may exert unexpected toxicity. The aim was therefore to compare toxicity of low doses of Se nanoparticles and the dissolved, ionized Se species selenite. Female rats were dosed orally for 28 d with either: 0.05, 0.5, or 4 mg Se/kg body weight (bw)/day as 20 nm Se nanoparticles or 0.05 or 0.5 mg Se/kg bw/day as sodium selenite. Male rats were dosed 4 mg Se/kg bw/day as Se nanoparticles. Body weight and clinical appearance were recorded throughout the experiment. At necropsy, blood samples were taken for hematological and clinical chemistry analyses; organ weights were recorded. At the high-dose of Se nanoparticles, overt toxicity occurred and the female animals had to be euthanized prematurely, whereas the male animals were reduced in dose. At all doses of Se nanoparticles and at 0.5 mg Se/kg bw/day as selenite, a lower body weight gain as compared to vehicle occurred. Relative liver weight was increased for both Se formulations at 0.5 mg Se/kg bw/day. Creatinine clearance and urinary pH were affected in some Se dosed groups. There were no effects among dosed groups on brain neurotransmitters or on hematological parameters compared with controls. There were no histological changes in the livers of animals exposed to Se nanoparticles or to selenite. Based on effects on body weight and liver weight, selenium nanoparticles and ionic Se exerted similar toxicity. This suggests that a nanoparticle-specific toxicity of Se did not occur.

Safety of orthosilicic acid-vanillin complex (OSA-VC) as a novel food ingredient to be used in food supplements as a source of silicon and bioavailability of silicon from the source.

The present scientific opinion deals with the safety of orthosilicic acid-vanillin complex (OSA-VC) as a novel food ingredient for use as a source of silicon (Si) in food supplements and with the bioavailability of Si from this source. OSA-VC is stable in liquid solution at low pH values. OSA from OSA-VC was available as revealed by the increase in plasma Si concentrations after oral ingestion in human volunteers. The toxicological data provided in support of the current application were not in accordance with the Tier 1 requirement of the 'Guidance for submission for food additive evaluations'; however, this was considered justified by the Panel given that OSA-VC at pH 6.8 dissociates into orthosilicic acid and vanillin. The daily consumption of OSA-VC at the dose recommended by the applicant would provide a supplemental intake of Si of approximately 10-18 mg Si/day which would result in an estimated total intake of roughly 30-70 mg Si/day. The maximum vanillin intake resulting from the consumption of OSA-VC would be less than 5% of the acceptable daily intake (ADI) value for vanillin of 10 mg/kg body weight (bw) per day established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2002. The Panel concluded that there would be no safety concern with the proposed use and use level of OSA-VC as a novel food ingredient intended to be used as a source of Si in food supplements for the adult population. The Panel concluded that OSA, measured as Si, is bioavailable following ingestion of OSA-VC and appears similar to values reported in the literature for other established sources of OSA.

Evaluation of di-calcium malate, used as a novel food ingredient and as a source of calcium in foods for the general population, food supplements, total diet replacement for weight control and food for special medical purposes.

The present scientific opinion deals with the evaluation of the safety of di-calcium malate (DCM) proposed as a novel food ingredient and as a source of calcium for use in foods for the general population, food supplements, total diet replacement for weight control and food for special medical purposes (FSMP), and with the bioavailability of calcium from this source. The structural formula of the proposed complex is based on expert judgement and not supported by any analytical data. On the basis of the available data, the Panel concluded that there was insufficient scientific evidence of a difference between the proposed novel food ingredient named as di-calcium malate (DCM) and calcium malate already authorised as a source of calcium included in Annex II to Directive 2002/46/EC. Accordingly, the Panel was unable to assess the safety of DCM as a novel food ingredient. On the basis of the results provided, the Panel considered that DCM does not completely dissociate into calcium and malic acid. The Panel concluded that when DCM dissociates, calcium would be available following ingestion of DCM and the bioavailability would appear similar to values reported for other sources of calcium already permitted. Furthermore, the Panel concluded that on the basis of the information available it was not possible to calculate the exposure to DCM as a source of calcium to foods for the general population, food supplements, total diet replacement for weight control and FSMP.

Evaluation of di-magnesium malate, used as a novel food ingredient and as a source of magnesium in foods for the general population, food supplements, total diet replacement for weight control and food for special medical purposes.

The present scientific opinion deals with the evaluation of the safety of di-magnesium malate (DMM) proposed as a novel food ingredient and as a source of magnesium for use in foods for the general population, food supplements, total diet replacement for weight control and food for special medical purposes (FSMP), and with the bioavailability of magnesium from this source. Additional information was sought from the applicant during the assessment process. However, despite several requests, the applicant did not provide the additional data. Consequently, the Panel performed this assessment on the basis of the available data and concluded that there was insufficient scientific evidence of a difference between the proposed novel food ingredient named as DMM and magnesium malate already authorised as a source of magnesium included in Annex II to Directive 2002/46/EC. Accordingly, the Panel was unable to assess the safety of DMM as a novel food ingredient. The Panel concluded that based on the data provided it was not possible to assess the dissociation of DMM into magnesium and malic acid. The Panel further concluded that if DMM dissociates, magnesium would be available following ingestion of DMM and the availability would appear similar to values reported for other sources of magnesium already permitted. Finally, the Panel noted that the proposed use levels could result in exposures to magnesium greater than its upper level (UL) (250 mg/day) for food supplements and for food for special medical purposes.

Guidance on safety evaluation of sources of nutrients and bioavailability of nutrient from the sources.

Whenever new substances are proposed for use as sources of nutrients in food supplements, foods for the general population or foods for specific groups, EFSA is requested by the European Commission to perform an assessment of their safety and of the bioavailability of the nutrient from the proposed source. This guidance describes the scientific data required to allow an evaluation of the safety of the source within the established framework for risk assessment of food additives and novel food ingredients and the bioavailability of the nutrient from this source. This document is arranged in five main sections: one on technical data aimed at characterising the proposed source and at identifying potential hazards resulting from its manufacture and stability in food; one on existing authorisations and evaluation, providing an overview of previous assessments on the proposed source and their conclusions; one on proposed uses and exposure assessment section, allowing an estimate of the dietary exposure to the source and the nutrient based on the proposed uses and use levels; one on toxicological data, describing approaches which can be used to identify (in conjunction with data on manufacture and composition) and to characterise hazards of the source and any relevant breakdown products; the final section on bioavailability focuses on determining the extent to which the nutrient from the proposed source is available for use by the body in comparison with one or more forms of the same nutrient that are already permitted for use on the positive lists. This guidance document should replace the previous guidance issued by the Scientific Committee for Food and published in 2001.

Re-evaluation of calcium silicate (E 552), magnesium silicate (E 553a(i)), magnesium trisilicate (E 553a(ii)) and talc (E 553b) as food additives.

The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re-evaluating the safety of calcium silicate (E 552), magnesium silicate (E 553a) and talc (E 553b) when used as food additives. In 1991, the Scientific Committee on Food (SCF) established a group acceptable daily intake (ADI) 'not specified' for silicon dioxide and silicates. The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) recently provided a scientific opinion re-evaluating the safety of silicon dioxide (E 551) when used as a food additive. The Panel noted that the absorption of silicates and talc was very low; there was no indication for genotoxicity or developmental toxicity for calcium and magnesium silicate and talc; and no confirmed cases of kidney effects have been found in the EudraVigilance database despite the wide and long-term use of high doses of magnesium trisilicate up to 4 g/person per day over decades. However, the Panel considered that accumulation of silicon from calcium silicate in the kidney and liver was reported in rats, and reliable data on subchronic and chronic toxicity, carcinogenicity and reproductive toxicity of silicates and talc were lacking. Therefore, the Panel concluded that the safety of calcium silicate (E 552), magnesium silicate (E 553a(i)), magnesium trisilicate (E 553a(ii)) and talc (E 553b) when used as food additives cannot be assessed. The Panel considered that there is no mechanistic rationale for a group ADI for silicates and silicon dioxide and the group ADI established by the SCF is obsolete. Based on the food supplement scenario considered as the most representative for risk characterisation, exposure to silicates (E 552-553) for all population groups was below the maximum daily dose of magnesium trisilicate used as an antacid (4 g/person per day). The Panel noted that there were a number of approaches, which could decrease the uncertainties in the current toxicological database. These approaches include - but are not limited to - toxicological studies as recommended for a Tier 1 approach as described in the EFSA Guidance for the submission of food additives and conducted with an adequately characterised material. Some recommendations for the revision of the EU specifications were proposed by the Panel.

High-fat feeding rather than obesity drives taxonomical and functional changes in the gut microbiota in mice.

BACKGROUND: It is well known that the microbiota of high-fat (HF) diet-induced obese mice differs from that of lean mice, but to what extent, this difference reflects the obese state or the diet is unclear. To dissociate changes in the gut microbiota associated with high HF feeding from those associated with obesity, we took advantage of the different susceptibility of C57BL/6JBomTac (BL6) and 129S6/SvEvTac (Sv129) mice to diet-induced obesity and of their different responses to inhibition of cyclooxygenase (COX) activity, where inhibition of COX activity in BL6 mice prevents HF diet-induced obesity, but in Sv129 mice accentuates obesity. RESULTS: Using HiSeq-based whole genome sequencing, we identified taxonomic and functional differences in the gut microbiota of the two mouse strains fed regular low-fat or HF diets with or without supplementation with the COX-inhibitor, indomethacin. HF feeding rather than obesity development led to distinct changes in the gut microbiota. We observed a robust increase in alpha diversity, gene count, abundance of genera known to be butyrate producers, and abundance of genes involved in butyrate production in Sv129 mice compared to BL6 mice fed either a LF or a HF diet. Conversely, the abundance of genes involved in propionate metabolism, associated with increased energy harvest, was higher in BL6 mice than Sv129 mice. CONCLUSIONS: The changes in the composition of the gut microbiota were predominantly driven by high-fat feeding rather than reflecting the obese state of the mice. Differences in the abundance of butyrate and propionate producing bacteria in the gut may at least in part contribute to the observed differences in obesity propensity in Sv129 and BL6 mice.

Acute and subacute pulmonary toxicity and mortality in mice after intratracheal instillation of ZnO nanoparticles in three laboratories.

Inhalation is the main pathway of ZnO exposure in the occupational environment but only few studies have addressed toxic effects after pulmonary exposure to ZnO nanoparticles (NP). Here we present results from three studies of pulmonary exposure and toxicity of ZnO NP in mice. The studies were prematurely terminated because interim results unexpectedly showed severe pulmonary toxicity. High bolus doses of ZnO NP (25 up to 100 µg; ≥1.4 mg/kg) were clearly associated with a dose dependent mortality in the mice. Lower doses (≥6 µg; ≥0.3 mg/kg) elicited acute toxicity in terms of reduced weight gain, desquamation of epithelial cells with concomitantly increased barrier permeability of the alveolar/blood as well as DNA damage. Oxidative stress was shown via a strong increase in lipid peroxidation and reduced glutathione in the pulmonary tissue. Two months post-exposure revealed no obvious toxicity for 12.5 and 25 µg on a range of parameters. However, mice that survived a high dose (50 µg; 2.7 mg/kg) had an increased pulmonary collagen accumulation (fibrosis) at a similar level as a high bolus dose of crystalline silica. The recovery from these toxicological effects appeared dose-dependent. The results indicate that alveolar deposition of ZnO NP may cause significant adverse health effects.

Absorption, distribution, metabolism and excretion of selenium following oral administration of elemental selenium nanoparticles or selenite in rats.

A suspension of nanoparticles of BSA-stabilized red amorphous elemental selenium (Se) or an aqueous solution of sodium selenite was repeatedly administered by oral gavage for 28 days at 0.05 mg kg(-1) bw per day (low dose) or at 0.5 mg kg(-1) bw per day (high dose) as Se to female rats. Prior to administration, the size distribution of the Se nanoparticles was characterized by dynamic light scattering and transmission electron microscopy, which showed that the particles' mean diameter was 19 nm and ranged in size from 10 to 80 nm. Following administration of the high dose of Se nanoparticles or selenite the concentration of Se was determined by ICP-MS in the liver, kidney, urine, feces, stomach, lungs, and plasma at the µg g(-1) level and in brain and muscle tissue at the sub-µg g(-1) level. In order to test if any elemental Se was present in the liver, kidney or feces, an in situ derivatization selective to elemental Se was performed by treatment with sulfite, which resulted in formation of the selenosulfate anion. This Se species was selectively and quantitatively determined by anion exchange HPLC and ICP-MS detection. The results showed that elemental Se was present in the livers, kidneys and feces of animals exposed to low and high doses of elemental Se nanoparticles or to selenite, and was also detected in the same samples from control animals. The fraction of Se present as elemental Se in livers and kidneys from the high dose animals was significantly larger than the similar fraction in samples from the low dose animals or from the controls. This suggested that the natural metabolic pathways of Se were exhausted when given the high dose of elemental Se or selenite resulting in a non-metabolized pool of elemental Se. Both dosage forms of Se were bioavailable as demonstrated by the blood biomarker selenoprotein P, which was equally up-regulated in the high-dose animals for both dosage forms of Se. Finally, the excretion of Se in urine and its occurrence as Se-methylseleno-N-acetyl-galactosamine and the trimethylselenonium-ion demonstrated that both dosage forms were metabolized and excreted. The results of the study showed that both forms of Se were equally absorbed, distributed, metabolized and excreted, but the detailed mechanism of the fate of the administered elemental Se or selenite in the gastro-intestinal tract of rats remains unclear.

Effect of rapeseed oil-derived plant sterol and stanol esters on atherosclerosis parameters in cholesterol-challenged heterozygous Watanabe heritable hyperlipidaemic rabbits.

Rapeseed oil (RSO) is a novel source of plant sterols, containing the unique brassicasterol in concentrations higher than allowed for plant sterol blends in food products in the European Union. Effects of RSO sterols and stanols on aortic atherosclerosis were studied in cholesterol-fed heterozygous Watanabe heritable hyperlipidaemic (Hh-WHHL) rabbits. Four groups (n 18 per group) received a cholesterol-added (2 g/kg) standard chow or this diet with added RSO stanol esters (17 g/kg), RSO stanol esters (34 g/kg) or RSO sterol esters (34 g/kg) for 18 weeks. Feeding RSO stanol esters increased plasma campestanol (P < 0.001) and sitostanol (P < 0.001) and aortic campestanol (P < 0.05) compared with controls. Feeding RSO sterol esters increased concentrations of plasma campesterol (P < 0.001), sitosterol (P < 0.001) and brassicasterol (P < 0.001) and aortic campesterol (P < 0.01). Significantly lower plasma cholesterol (P < 0.001) was recorded in the treated groups after 3 weeks and throughout the study. LDL-cholesterol was reduced 50 % in the high-dose RSO sterol ester (P < 0.01) and high-dose RSO stanol ester (P < 0.001) groups compared with controls. Atherosclerotic lesions were found in three rabbits in each of the RSO stanol ester groups and in one in the RSO sterol ester group. Aortic cholesterol was decreased in the treated groups (P < 0.001) in response to lowering of plasma cholesterol induced by RSO sterol and stanol esters. In conclusion, RSO stanol and sterol esters with a high concentration of brassicasterol were well tolerated. They were hypocholesterolaemic and inhibited experimental atherosclerosis in cholesterol-fed Hh-WHHL rabbits. A significant uptake of plant sterols into the blood and incorporation of campesterol and campestanol into aortic tissue was recorded.

Increased plant sterol and stanol levels in brain of Watanabe rabbits fed rapeseed oil derived plant sterol or stanol esters.

Foods containing plant sterol or stanol esters can be beneficial in lowering LDL-cholesterol concentration, a major risk factor for CVD. The present study examined whether high dietary intake of rapeseed oil (RSO) derived plant sterol and stanol esters is associated with increased levels of these components in brain tissue of homozygous and heterozygous Watanabe rabbits, an animal model for familial hypercholesterolemia. Homozygous animals received either a standard diet, RSO stanol or RSO sterol ester while heterozygous animals were additionally fed with 2 g cholesterol/kg to the respective diet form for 120 d (n 9 for each group). Concentrations of cholesterol, its precursor lathosterol, plant sterols and stanols in brain and additionally in liver and plasma were determined by highly sensitive GC-MS. High-dose intake of RSO derived plant sterols and stanols resulted in increased levels of these components in plasma and liver. In brain a limited uptake of plant sterols and stanols was proven, indicating that these compounds passed the blood-brain barrier and may be retained in the brain tissue of Watanabe rabbits. Plant stanol ester feeding lowered plant sterol levels in brain, liver, and plasma. Cholesterol synthesis in brain, indicated by lathosterol, a local surrogate cholesterol synthesis marker, does not seem to be affected by plant sterol or stanol ester feeding. We conclude that high dose intake of plant sterol and stanol esters in Watanabe rabbits results in elevated concentrations of these components not only in the periphery but also in the central nervous system.

Effects of red grape skin and seed extract supplementation on atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

Epidemiological studies have suggested an association between consumption of red wine and other polyphenolic compounds and prevention of cardiovascular diseases. In the present study, Watanabe heritable hyperlipidemic (WHHL) rabbits were used to investigate the effects of polyphenols in a red grape skin and seed extract (GSE) on the development of atherosclerosis. WHHL rabbits received either semisynthetic diet (casein based) or semisynthetic diet added GSE over a period of 15 wk. Plasma lipids and aortic cholesterol accumulation were measured. Feeding semisynthetic diet was associated with increasing hypercholesterolemia, which was developing slower in GSE group compared to the controls as recorded by significantly lower plasma cholesterol in dosage week 7 (males: P < 0.05, females: P < 0.01) and 11 (males: P < 0.01). Aortic atherosclerosis evaluated as the cholesterol content in aortic tissue was comparable in the control and GSE-dosed females, but it was significantly reduced in the abdominal part of GSE-dosed male compared to the controls (P < 0.05). In conclusion, feeding GSE extract to WHHL rabbits had no significant effects in females but was associated with transient less hypercholesterolemic response to semisynthetic diet and, furthermore, retarded the development of aortic atherosclerosis in males as demonstrated by significantly lower cholesterol content in the abdominal part.

Dietary supplementation with an extract of lycopene-rich tomatoes does not reduce atherosclerosis in Watanabe Heritable Hyperlipidemic rabbits.

Tomatoes are rich in lycopene and other carotenoids which have shown beneficial effects on CVD in epidemiological and intervention studies. In the present study the effect of an extract of lycopene-rich tomatoes, Lyc-O-Mato on atherosclerosis was studies in Watanabe Heritable Hyperlipidemic rabbits. The rabbits were fed a control diet, a control diet supplemented with the tomato extract or a control diet supplemented with a mixture of plant oils for 16 weeks. Lycopene was detected only in plasma of rabbits receiving tomato extract. The tomato extract had no effect on cholesterol and triacylglycerol levels measured in total plasma, lipoprotein fractions and on aortic atherosclerosis evaluated biochemically and by microscopy. Oxidation of lipids in unfractionated plasma also was unaffected by the intake of tomato extract. In conclusion, the tomato extract increased plasma levels of lycopene in rabbits, but had no effect on hypercholesterolaemia, oxidation of plasma lipids or aortic atherosclerosis.

Estrogen and phytoestrogens: Effect on eNOS expression and in vitro vasodilation in cerebral arteries in ovariectomized Watanabe heritable hyperlipidemic rabbits.

OBJECTIVES: To evaluate the effect of estrogen replacement therapy or soy isoflavones supplement on endothelium-dependent relaxation in vitro and gene expression of endothelial nitric oxide synthase (eNOS) in cerebral arteries in a rabbit model of human hypercholesterolemia. STUDY DESIGN: Thirty-six female ovariectomized Watanabe heritable hyperlipidemic (WHHL) rabbits were randomised to treatment with 17beta-estradiol (17beta-E(2)), SoyLife 150 or control for 16 weeks. Ring segments of basilar artery (BA) and posterior cerebral artery (PCA) were mounted in myographs for isometric tension recordings. Concentration response curves to carbamylcholine chloride, sodium nitroprusside (SNP) and l-NAME were evaluated after precontraction with potassium. Total RNA was extracted, reverse transcribed and eNOS quantitated by real-time polymerase chain reaction (real-time PCR). RESULTS: Plasma cholesterol was significantly higher at termination in the SoyLife group (P<0.0001), whereas low-density lipoprotein (LDL) cholesterol was comparable in all treatment groups. Neither treatment influenced the endothelium-dependent responses to carbamylcholine chloride or l-NAME or the endothelium-independent response to SNP in any of the arteries. Correspondingly, eNOS mRNA was similarly expressed in all treatment groups in both arteries. CONCLUSIONS: Improvement of cerebral endothelial function by estrogen or soy isoflavones in ovariectomized WHHL rabbits is not supported by the present data. The findings may be unique to the WHHL rabbit in which the hypocholesterolemic effect of estrogens mediated by upregulation of liver LDL receptors is excluded.

Tissue lipid lowering-effect of a traditional Nigerian anti-diabetic infusion of Rauwolfia vomitoria foilage and Citrus aurantium fruit.

The toxicity and anti-diabetic properties of an aqueous plant extract made by boiling Rauwolfia vomitoria foilage and Citrus aurantium fruits were evaluated in mice. A single dosage corresponding to 70x the human-daily-dose was non-toxic when administered to 6-week-old NMRI lean mice or 6- or 11-week-old C57BL/6J lean mice. Daily treatment of 11-week-old C57BL/KsBom-db (db/db) genetic diabetic mice with a dose corresponding to 10x human-daily-dose for 6 weeks facilitated a significant weight loss as compared to the untreated controls. During treatment, the db/db mice were maintained on the carbohydrate-deficient Altromin C1009 diet. Although the food intake in the treated mice was not statistically significant from that in the controls, the treated animals had significantly higher serum triglyceride contents, suggesting that the treatment induced lipid mobilization from internal stores. Moreover, the fatty acid profile of the eyes from the treated animals showed a significant reduction in total fatty acid content accompanied by a 33% reduction in estimated Stearoyl-CoA desaturase activity (p = 0.039) as compared with controls. The fatty acid mobilization and a protection of the brittle C57BL/KsBom-db pancreas were observed 5 weeks after cessation of treatment when the treated animals were maintained on the poorer Altromin C1009 diet.