Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety

Premarket, genetically modified (GM) plants are assessed for potential risks of food allergy. The major risk would be transfer of a gene encoding an allergen or protein nearly identical to an allergen into a different food source, which can be assessed by specific serum testing. The potential that a newly expressed protein might become an allergen is evaluated based on resistance to digestion in pepsin and abundance in food fractions. If the modified plant is a common allergenic source (e.g. soybean), regulatory guidelines suggest testing for increases in the expression of endogenous allergens. Some regulators request evaluating endogenous allergens for rarely allergenic plants (e.g. maize and rice). Since allergic individuals must avoid foods containing their allergen (e.g. peanut, soybean, maize, or rice), the relevance of the tests is unclear. Furthermore, no acceptance criteria are established and little is known about the natural variation in allergen concentrations in these crops. Our results demonstrate a 15‐fold difference in the major maize allergen, lipid transfer protein between nine varieties, and complex variation in IgE binding to various soybean varieties. We question the value of evaluating endogenous allergens in GM plants unless the intent of the modification was production of a hypoallergenic crop.     

A new immunomodulatory function of low-viscous konjac glucomannan with a small particle size: its oral intake suppresses spontaneously occurring dermatitis in NC/Nga mice

BACKGROUND: Konjac glucomannan (GM) is a well-known dietary fiber with various beneficial functions: the higher viscosity displayed the stronger potency. However, the high-viscous GM powders, ordinary konjac powder and highly purified GM were mostly unsuitable for the application to various food industries. Our aims are to develop new physiological functions of low-viscous GM powder, pulverized GM or re-granulated fine GM, using a murine model of atopic dermatitis. METHODS: Male 4-week-old NC/Nga mice were fed for 8 weeks on diets containing 5% of two high-viscous and two low-viscous GM powders, respectively. RESULTS: Striking suppression against the aggravation of dermatitis, the increase in scratching behaviors, and the rise in IgE levels was recognized only in mice fed on the pulverized GM diet, but not in mice fed on the other GM diets or a control diet. Eczema prevention in the fine GM-fed mice was accompanied by a significant decrease in their plasma IFN-gamma levels, a positive regulatory cytokine for atopic skin inflammation. CONCLUSION: Only the pulverized GM possessed the ability to suppress the development of dermatitis in NC/Nga mice. This is the new immunomodulatory function of low-viscous GM with a small particle size.     

Evaluating the allergic risk of genetically modified soybean

Genetically modified (GM) soybean (carrying the EPSPS transgene) is the most common GM food in Korea. In order to assess whether genetic modification increases the allergenic risk of soybeans, the allergenicity and IgE-reactive components of wild-type and GM soybean extracts were compared in allergic adults who had been sensitized to soybeans. We enrolled 1,716 adult allergy patients and 40 healthy, non-atopic controls. Skin prick tests and IgE enzyme linked immunosorbent assays (ELISAs) were performed using wild-type and GM soybean extracts, along with other common inhaled allergens. The specificities of serum IgE antibodies from allergic patients and the identities of the IgE-reactive components of the soybean extracts were compared using ELISA inhibition testing, 2-dimensional gel electrophoresis, and IgE immunoblotting. To evaluate the effects of digestive enzymes and heat treatment, the soybean extracts were heated or pre- incubated with or without simulated gastric and intestinal fluids. The IgE sensitization rates to wild-type and GM soybeans were identical (3.8% of allergic adults), and circulating IgE antibodies specific for the two extracts were comparable. The results of the ELISA inhibition test, SDS-PAGE, and IgE immunoblotting showed a similar composition of IgE-binding components within the wild-type and GM extracts, which was confirmed using two-dimensional gel electrophoresis, IgE immunoblotting, and amino acid sequencing. None of the subjects had a positive response to purified EPSPS protein in the skin prick test, ELISA, or IgE immunoblot analysis. These findings suggest that the IgE sensitization rate to GM soybean extracts is identical to that of wild-type soybean extracts in adult allergy patients. In addition, based on both in vivo and in vitro methods, the allergenicity of wild type and GM soybean extracts was identical.     

Assessing genetically modified crops to minimize the risk of increased food allergy: a review

The first genetically modified (GM) crops approved for food use (tomato and soybean) were evaluated for safety by the United States Food and Drug Administration prior to commercial production. Among other factors, those products and all additional GM crops that have been grown commercially have been evaluated for potential increases in allergenic properties using methods that are consistent with the current understanding of food allergens and knowledge regarding the prediction of allergenic activity. Although there have been refinements, the key aspects of the evaluation have not changed. The allergenic properties of the gene donor and the host (recipient) organisms are considered in determining the appropriate testing strategy. The amino acid sequence of the encoded protein is compared to all known allergens to determine whether the protein is a known allergen or is sufficiently similar to any known allergen to indicate an increased probability of allergic cross-reactivity. Stability of the protein in the presence of acid with the stomach protease pepsin is tested as a risk factor for food allergenicity. In vitro or in vivo human IgE binding are tested when appropriate, if the gene donor is an allergen or the sequence of the protein is similar to an allergen. Serum donors and skin test subjects are selected based on their proven allergic responses to the gene donor or to material containing the allergen that was matched in sequence. While some scientists and regulators have suggested using animal models, performing broadly targeted serum IgE testing or extensive pre- or post-market clinical tests, current evidence does not support these tests as being predictive or practical. Based on the evidence to date, the current assessment process has worked well to prevent the unintended introduction of allergens in commercial GM crops.     

Lipid peroxidation in rat brain is increased by simulated weightlessness and decreased by a soy-protein diet

This study tested whether or not simulated weightlessness by tail-suspension increases the levels of lipid peroxidation products in rat brain. The brain tissues of rats on a soybean diet were also assayed for lipid peroxidation products to evaluate the possible role of soy-protein as a dietary anti-oxidant. Male Sprague-Dawley rats were used. Group 1 rats were fed standard Purina rat chow ad libidum and served as controls. Group 2 rats were fed a soybean diet containing 37% soy-protein and were not tail-suspended. Group 3 rats were fed standard Purina rat chow ad libidum and were tail-suspended to induce simulated weightlessness. After 2 wk, all of the rats were killed. Each whole brain was segmented into frontal cortex, cerebellum, and brain stem. After a specific weight of each segment was excised, the residual tissues were combined and used as a whole brain sample. The samples were analyzed for lipid peroxidation products by a chromogenic assay that reacts with malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE). The mean concentrations of lipid peroxidation products (MDA plus 4-HNE) in whole brain, frontal cortex, cerebellum, and brain stem of the control rats ranged from 16 to 18 micromol/g; the corresponding means ranged from 10 to 13 micromol/g in rats fed the soybean diet, and from 22 to 26 micromol/g in the tail-suspended rats. Thus, the mean levels of lipid peroxidation products in brain tissues were decreased in the rats fed the soy diet and were increased in the rats that were tail-suspended to simulate weightlessness, when compared to those of rats fed a regular diet.     

Hepatoprotective properties of soybean proteins and their use for dietary therapy of chronic toxic hepatitis

Hepatoprotective effect of a diet containing soybean proteins most pronounced in soybean milk was demonstrated on the model of chronic toxic hepatitis. Results of biochemical studies correlate with morphological data indicating that soybean proteins hold much promise for supportive therapy of patients with chronic liver diseases. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 3, pp. 283–286, March, 2000     

Effect of soybean meal fermented with Bacillus subtilis BS12 on growth performance and small intestinal immune status of piglets

This study was aimed to screen a high-efficient strain for degrading antigenic protein in soybean meal (SBM) and evaluate the effect of fermented soybean meal (FSBM) on growth performance of piglets. A Bacillus subtilis strain BS12 was selected through plate and fermentation experiment, which reduced 92.36% less glycinin and 88.44% less β-conglycinin in SBM. A total of 192 piglets were assigned to receive either a diet of SBM with antibiotics (the control group) or a diet containing 10% FSBM without antibiotics. The average daily gain and feed intake of pigs fed FSBM were superior (p?p?相似文献   

Voluntary consumption of ethyl oleate reduces food intake and body weight in rats

Previous studies have shown that administration of the fatty acids, linoleic and oleic acid, either by intragastric or intraintestinal infusion, suppresses food intake and body weight in rats. While still not fully understood, gut-mediated satiety mechanisms likely are potential effectors of this robust response to gastrointestinal fatty acid infusions. The objective of this study was to assess the effects of voluntary access to an oleic acid derivative, ethyl oleate (EO), on subsequent food intake and body weight in rats. Animals were randomized either to a 12.5% EO diet or a soybean oil diet as a "breakfast," followed either by two one-hour or one five-hour access periods to standard rodent diet, and food intake and body weights were collected. Across 14 days access, rats consuming EO on both feeding schedules gained less weight and consumed less total kilocalories than rats consuming the SO diet. Further, plasma levels of glucose and insulin were comparable in both EO and SO diet groups. In summary, EO was found to increase weight loss in rats maintained on a 75% food-restriction regimen, and attenuate weight-gain upon resumption of an ad-libitum feeding regimen. These data indicate that voluntary access to EO promoted short-term satiety, compared to SO diet, and that these effects contributed to an important and novel attenuated weight gain in EO-fed animals.     

Variation of fat intake with estrous cycle, ovariectomy and estradiol replacement in hamsters (Mesocricetus auratus) eating a fractionated diet

Intakes of fats, proteins, and carbohydrates were measured daily for a 3 month period in female hamsters maintained on a fractionated diet consisting of the dietary constituents, dextrose, fat and soybean meal. In the first study, hamsters showed variations in body weight and fat intake across the estrous cycle and following ovariectomy, with elevations during diestrous and after ovariectomy when endogenous estrogens are reduced. In the second study, the effects of exogenous estradiol or cholesterol on intake patterns of ovariectomized hamsters were determined. Hamsters lost weight and decreased fat intake when implanted with a silastic containing estradiol and gained weight and increased fat intake when exposed to cholesterol treatment. In neither of the two studies were the intakes of protein or carbohydrates significantly affected by the animals' hormonal status. These results suggest that in the hamster, estrogenic effects on food intake are specific to some dietary constituents and not to others.     

Dairy milk fat augments paclitaxel therapy to suppress tumour metastasis in mice,and protects against the side-effects of chemotherapy

Milk fat is a natural product containing essential nutrients as well as fatty acids and other food factors with reported anti-cancer potential. Here bovine milk fat was tested for its ability to inhibit the growth of breast and colon cancers and their metastasis to the lung and liver; either alone or in combination with the chemotherapeutic agent paclitaxel. A diet containing 5% typical anhydrous milk fat (representing ~70% of the total dietary fat component) fed to Balb/c mice delayed the appearance of subcutaneous 4T1 breast and CT26 colon cancer tumours and inhibited their metastasis to the lung and liver, when compared to the control diet containing soybean oil as the only fat component. It augmented the inhibitory effects of paclitaxel on tumour growth and metastasis, and reduced the microvessel density of tumours. It displayed no apparent organ toxicity, but instead was beneficial for well-being of tumour-bearing mice by maintaining gastrocnemius muscle and epididymal adipose tissue that were otherwise depleted by cachexia. The milk fat diet ameliorated gut damage caused by paclitaxel in non-tumour-bearing mice, as evidenced by retention of jejunal morphology, villi length and intestinal γ-glutamyl transpeptidase activity, and inhibition of crypt apoptosis. It prevented loss of red and white blood cells due to both cancer-mediated immunosuppression and the cytotoxic effects of chemotherapy. The present study warrants the use of milk fat as an adjuvant to inhibit tumour metastasis during cancer chemotherapy, and to spare patients from the debilitating side-effects of cytotoxic drugs.     

Influence of experience on response to bitter taste

Sucrose octaacetate, which tastes bitter to humans, can reduce the energy intake of rats when added to their diet. The reduction in energy intake is transient, lasting no more than 5 weeks. Rats that no longer reduce intake in response to sucrose octaacetate still avoid food containing it in choice tests, although to a lesser degree than rats having no previous experience with sucrose octaacetate. The ability of sucrose octaacetate to reduce preference without reducing long-term intake accounts for the previous finding that a wet diet containing sucrose octaacetate can stimulate hyperphagia yet be less preferred than the control diet. Sucrose octaacetate was more effective in reducing intake of a wet than of a dry diet. This last observation indicates that adding water to a food makes it easier for an animal to taste its food.     

Effects of hippocampal lesions on adaptive intake of diets with disproportionate amounts of amino acids

Bilateral double electrolytic overlapping lesions were placed in dorsal-lateral hippocampus of male 230 g rats, and their food intake responses to the ingestion of diets containing disproportionate amounts of amino acids were examined. Rats with such lesions and intact control rats maintained their normal intakes of the 6% casein basal diet or a threonine basal amino acid diet postoperatively. However, they exhibited marked initial food intake depression, similar to that of intact rats, when fed the threonine imbalanced amino acid diet. Also, animals with lesions in certain areas of the dorsal-lateral hippocampus showed facilitated adaptation to the amino acid imbalanced diet. Similar severe reduction in food intake with relative lack of adaptation were observed in both the intact controls and rats with hippocampal lesions when fed amino acid diets completely devoid of threonine. Initial food intake of rats with hippocampal lesions was inhibited drastically as was the case with the intact controls when fed a 75% casein high protein diet. All rats, either intact or lesioned, showed similar slow adaptation patterns with the prolonged ingestion of the high protein diet. The initial food intake responses and facilitated adaptation of the animals bearing lesions in certain areas of the hippocampus suggest that such areas are not crucially involved in the inhibition of food intake of rats fed disproportionate amounts of dietary amino acids. Rather, such areas of lesions in the hippocampus may play a role in a system governing the behavioral adaptation of the intake of amino acid imbalanced diets but not of diets containing amino acids in general excess. This would also indicate that different mechanisms control the intake of amino acid imbalanced diets and diets containing amino acids in excess.     

Effects of dietary protein on food and water intake in spontaneously hypertensive rats

We have been studying the development of hypertension in spontaneously hypertensive rats (SHR) fed a low protein diet. The effects of a low protein diet upon food and water intake were examined. Body weight gain, food and water intake were measured in three to twenty-three week-old SHR and Wistar Kyoto rats (WKY) fed diets containing 8%, 15% or 25% casein. Body weights of SHR and WKY fed an 8% casein diet were significantly lower at 23 weeks than rats on the higher protein diets, although both groups on the 8% diet consumed more food and water per g of body weight. In addition, SHR fed an 8% casein diet drank less water per gram of food than WKY or SHR fed 15% and 25% casein diets. These results indicate that changes in food and water intake, as a consequence of low protein diets, should be an additional consideration when examining the effects of dietary protein on the development of hypertension.     

Caloric regulation in normal-weight men maintained on a palatable diet of concentional foods

The spontaneous food intake of six normal-weight male volunteers was measured for 24 days while the subjects were inpatients on a metabolic unit. They were fed a palatable diet of conventional foods and were kept unaware that their food intake was being measured. On days 7–18 the caloric content of their diet was covertly reduced by 25% by substituting aspartame-sweetened analogues for all menu items containing sucrose. Subjects did not alter their food intake for 3 days. Then between days 4–6 on the aspartame diet, they increased their intake to compensate for 40% of the missing calories. Food intake stabilized at 85% of baseline and remained the same for the rest of the 12-day dilution period. Subjects did not show a shift in either sweetened or unsweetened food choices while their diet was being diluted. In adjusting for the missing calories, they simply ate more of their customary diet. The replacement of sucrose by aspartame tended to curb the weight gain observed on the baseline diet.     

Alleviation in the rat of a GABA-induced reduction in food intake and growth

Cold exposure and diet dilution which stimulate food intake of normal rats lessened depressions of food intake and growth induced by dietary GABA. During a 3-day adaptation to the cold, rats fed a diet containing 4.5% GABA lost weight; thereafter, food intake and growth rate differed little from those of cold control rats and were usually greater than those of normal rats fed GABA. Hepatic GABA-aminotransferase activity of cold-exposed rats fed the GABA diet increased to about twice that of normal control rats. Rats fed a control diet diluted by half with cellulose ate 50% more of this diet than of the undiluted diet but gained only 20% less weight. Rats ate twice as much of a diluted, 9% GABA diet as of an undiluted, 4.5% GABA diet (thus doubling their GABA intake) and gained three times as much weight. A novel food (condensed milk) barely lessened the adverse responses to GABA. These results show that conditions requiring rats to increase their food intake in order to maintain body weight can also increase their acceptance of a diet high in GABA.     

Effects of dietary dehydroepiandrosterone on food intake and body weight in rats with medial hypothalamic knife cuts

Dehydroepiandrosterone (DHEA) is a steroid which has been reported to have anti-obesity effects when added to the diets of rats and mice. In this report, rats made hyperphagic with medial hypothalamic knife cuts were placed on a diet containing 0.45% DHEA or a control diet. Knife cut rats on the control diet ate more food and gained more weight than sham-operated rats on the control diet. In contrast, knife cut rats fed the DHEA diet weighed the same as shams on the DHEA diet and were only observed to be hyperphagic on one of eight 24 hour measurements taken during a five week period. Dietary DHEA reduced food intake and body weight of both knife cut and sham-operated rats, though the effects were smaller in shams. As these effects of DHEA were reminiscent of the effects of dietary quinine adulteration on intake by knife cut rats, a second experiment measured the food intake of unoperated rats when given a choice between a control high-fat diet and one adulterated with various concentrations of DHEA. Even at a concentration of 0.05%, rats clearly identified and avoided the DHEA-adulterated diet. While these results do not rule out effects of DHEA on metabolic rate or lipogenesis, they do indicate that the unpalatability of DHEA-adulterated diets may be a contributing factor in the observed effects on food intake and body weight.     

Myocardial ultrastructure of rats fed high and low erucic acid rapeseed oils

Ultrastructural study was conducted on ventricular muscle obtained from male rats of the Sprague-Dawley strain after 18 and 28 weeks of feeding diets containing 20% (w/w) soybean oil, low erucic acid rapeseed oil, or high erucic acid rapeseed oil. Myocardium of the rats fed soybean oil-containing diet was apparently normal despite high content of oil in the diet. Long-term feeding of high erucic acid rapeseed oil resulted in alteration of mitochondrial morphology, disorganization of myofibrils, and degeneration or necrosis of the cardiac muscle fibers. Macrophages were observed near the degenerating muscle cells but these cells did not exhibit accumulation of lipid droplets. Old lesions were characterized by an increase in collagen fibrils. Low erucic acid rapeseed oil induced less severe cardiopathologic changes but the nature of the alterations was similar to that of high erucic acid rapeseed oil. Presence of abnormal mitochondria in the cardiac muscle cells of the rats containing focal myocardial necrosis implies that transitions in mitochondrial structure, composition, and function may be responsible for the development of the focal myocardial lesions in rapeseed oil-fed rats.     

Safety assessment and public concern for genetically modified food products: the European view

The safety assessment for marketing purposes of genetically modified (GM) foods in the 15 Member States of the European Union (EU) is based on the Novel Foods and Novel Food Ingredients Regulation adopted in May 1997. Before a GM food can be approved under the Regulation, it must satisfy three criteria: Gm food must be safe, it must not mislead the consumer and it must be nutritionally adequate. The EU Scientific Committee on Food has published a set of guidelines describing the type of information expected from a company in support of an application for approval of a GM food or food ingredient. Despite this rigorous procedure and there being no evidence of harm resulting from the consumption of GM foods worldwide, there is essentially no market in the EU for such products at present. Possible reasons for this are discussed and the view put forward that the market for GM foods will change only when there are more clearly perceived consumer benefits.     

Hepatic vagotomy increases consumption of a novel-tasting diet presented immediately after surgery

To further characterize the possible effect of hepatic vagotomy on food intake, rats adapted to a medium-fat diet (MF diet) were offered a novel-tasting diet immediately after hepatic branch vagotomy (HBX rats) or sham vagotomy (SHAM rats) and food intake as well as diet selection (novel-tasting vs. familiar diet) was measured. When a novel-tasting sweet medium-fat diet (SMF diet) was offered immediately after surgery, HBX rats ate transiently more (days 1-4 after surgery) and selected more (days 5 and 6 after surgery) of the SMF diet than SHAM rats. Similar results were obtained when a novel-tasting bitter diet (AMF diet) was offered instead of the SMF diet. In contrast, when rats adapted to the AMF diet prior to surgery were given normal MF diet as novel-tasting diet immediately after surgery, HBX and SHAM rats' postoperative food intake and diet selection did not differ. When rats were given only MF or SMF diet before and after surgery, HBX and SHAM rats' food intake during the initial postoperative period also did not differ. The results indicate that hepatic branch vagotomy and sham vagotomy have different aversive effects which lead to transient differences in postoperative food intake when diets with a novel and strong taste are presented during the initial postoperative period. Therefore, information conveyed by the hepatic branch of the vagus can apparently lead to a conditioned feeding response. This is evidence for a more subtle role of the liver in the control of food intake than previously thought.     

Aversion of the cat to dietary medium-chain triglycerides and caprylic acid

Young, specific-pathogen-free cats were fed purified diets containing different sources of fat. Food intake was depressed and cats lost weight when the diet contained either hydrogenated coconut oil (HCO) or medium-chain triglycerides (MCT). With an MCT preparation enriched in 8:0 (MCT8), cats would not eat after first tasting the diet. When cats were offered a choice of two high-fat diets, they chose the basal diet over a diet containing 30% HCO, by a ratio of 4.5:1. Low levels of MCT8 (5% or 10% by weight) were also rejected, whereas cats did not reject 5% or 15% MCT12. Caprylic acid, at 0.1-1.0% of the diet, was rejected. In other studies, food intake and body weight decreased when HCO was added to a fat-free diet. Cats fed 25% or 35% HCO lost weight. When 5% safflower seed oil was added to the HCO diets, body weights and food intake improved, but were still less than optimal. These studies indicate that the food intake depression in cats fed dietary HCO and MCT is primarily a result of impalatability, and that the fatty acid moiety may be responsible for the aversion.