Genetic and acute toxicological evaluation of an algal oil containing eicosapentaenoic acid (EPA) and palmitoleic acid.

Algal strains of Nannochloropsis sp. were developed, optimized, cultivated and harvested to produce a unique composition of algal oil ethyl esters (Algal-EE) that are naturally high in eicosapentaenoic acid (EPA, 23-30%) and palmitoleic acid (20-25%), and contain no docosahexaenoic acid (DHA). Algal-EE was evaluated for mutagenic activity (Ames bacterial reverse mutation, in vitro mammalian chromosome aberration, in vivo micronucleus test) and for acute oral toxicity in Sprague-Dawley rats. In the acute toxicity study, rats received a single oral gavaged dose of Algal-EE (2000 mg/kg body weight). Clinical observations were made for 14 days before sacrifice on Day 15. Macroscopic evaluation involved the examination of all organs in the cranial, thoracic, and abdominal cavities. Algal-EE showed no evidence of mutagenicity, did not produce an increase in the frequency of structural chromosome aberrations, and did not cause an increase in the induction of micronucleated polychromatic erythrocytes. There were no macroscopic abnormalities. Algal-EE up to 2000 mg/kg body weight did not affect body weight, organ appearance or produce any toxic-related signs of morbidity. The acute median lethal dose (LD50) of Algal-EE was >2000 mg/kg body weight. Based on these assays, Algal-EE does not appear to have any genetic or acute oral toxicity.

Comparison of broiler performance when fed diets containing grain from YieldGard (MON810), YieldGard x Roundup Ready (GA21), nontransgenic control, or commercial corn.

This 42-day experiment was undertaken to compare the nutritional value of insect-protected corn event MON810 (YieldGard) and YieldGard x herbicide-tolerant corn event GA21 (Roundup Ready) to their nontransgenic controls as well as four different commercial reference corns, when fed to growing Cobb x Cobb broilers. A randomized complete block design was used, and each treatment was replicated with five pens of males and five pens of females with 10 broilers per pen. Broilers were fed approximately 55% wt/wt corn during the first 20 d and approximately 60% wt/wt corn thereafter. The corn component of diets fed to broilers was supplied entirely with grain from the eight hybrids included in the experiment. Final live weights averaged 2.09 kg/bird fed YieldGard corn and 2.15 kg/bird fed YieldGard x Roundup Ready corn and were not different (P > 0.05) from final weights for birds fed control or commercial corn. Feed conversion was not affected (P > 0.05) by YieldGard (1.72) or YieldGard x Roundup Ready (1.77) corn feeding when compared with the feeding of other corn diets. Chill weights, fat pad, thigh weights, and wing weights were not affected by diets (P > 0.05). Differences (P < 0.05) were noted for breast and drum weights across treatments. Broilers overall performed consistently and had similar carcass yield and meat composition when fed diets containing YieldGard (event MON810) or YieldGard (event MON810) x Roundup Ready (event GA21) as compared with their nontransgenic controls and commercial diets.

Comparison of broiler performance when fed diets containing grain from roundup ready (NK603), yieldgard x roundup ready (MON810 x NK603), non-transgenic control, or commercial corn.

Two 42-d experiments compared the nutritional value of the glyphosate-tolerant corn event NK603 (Roundup Ready corn) (experiment 1) and the combined traits, insect-protected corn event MON 810 (YieldGard com) x glyphosate-tolerant corn event NK603 (experiment 2) to their respective non-transgenic controls and to commercial reference corn, when fed to growing broilers. For each experiment, a randomized complete block design was used with eight dietary treatments in each of five replicated blocks of pens (eight pens for males and eight pens for females per block). Final live weights and feed conversion were not different (P > 0.05) across all treatments in both experiments. In experiment 1, broilers fed diets containing Roundup Ready corn had similar feed conversion adjusted for mortalities to those fed the non-transgenic control and one of the commercial corn diets. Chill weights and thigh, drum, and wing weights were not affected by diets. Differences (P < 0.05) were noted for breast meat and fat pad weights across treatments. In experiment 2, the adjusted feed conversion and carcass parameters were not affected by diets. Differences (P < 0.05) were noted only for protein content of breast meat. Differences observed in both experiments were consistent with natural variability. Broilers in general performed consistently and had similar carcass yields and meat compositions when fed diets containing Roundup Ready corn or YieldGard x Roundup Ready corn as compared with their respective non-transgenic control and commercial diets supporting similar feeding values among diets.

Comparison of broiler performance when fed diets containing grain from YieldGard Rootworm (MON863), YieldGard Plus (MON810 x MON863), nontransgenic control, or commercial reference corn hybrids.

Two 42-d experiments compared the nutritional value of YieldGard Rootworm corn (MON863; experiment 1) and YieldGard Plus corn (MON810 x MON863; experiment 2) to their respective nontransgenic controls and 6 commercial reference corn hybrids when fed to growing broilers. For each experiment, a randomized complete block design was used with 8 dietary treatments in each of 5 replicated blocks of pens. In experiment 1, no differences among diets were observed (P > 0.05) for final live weights and feed conversion. Broilers fed diets containing MON863 corn had adjusted feed conversion similar to the nontransgenic control and the population of control and commercial diets. On a weight basis, there were no differences among diets for chill, fat pad, and thigh, drum, and wing weights. Differences (P < 0.05) between MON863 and commercial corn diets were noted for breast meat, chill and thigh, drum, and wing weights on a percentage of weight basis. No differences were observed (P > 0.05) in the percentage of moisture, protein, and fat in breast meat or thigh meat across treatment diets. In experiment 2, there were no significant differences among diets for all broiler performance and carcass parameters evaluated. Broilers overall performed consistently and had similar carcass yields and meat compositions when fed diets containing MON863 corn or MON810 x MON863 corn as compared with their respective nontransgenic control and commercial diets, supporting a conclusion of similar feeding values among diets.

Isolation and characterization of cDNAs expressed in the early stages of flavonol-induced pollen germination in petunia.

Petunia (Petunia hybrida) pollen requires flavonols (Fl) to germinate. Adding kaempferol to Fl-deficient pollen causes rapid and synchronous germination and tube outgrowth. We exploited this system to identify genes responsive to Fls and to examine the changes in gene expression that occur during the first 0.5 h of pollen germination. We used a subtracted library and differential screening to identify 22 petunia germinating pollen clones. All but two were expressed exclusively in pollen and half of the clones were rare or low abundance cDNAs. RNA gel-blot analysis showed that the steady-state transcript levels of all the clones were increased in response to kaempferol. The sequences showing the greatest response to kaempferol encode proteins that have regulatory or signaling functions and include S/D4, a leucine-rich repeat protein, S/D1, a LIM-domain protein, and D14, a putative Zn finger protein with a heme-binding site. Eight of the clones were novel including S/D10, a cDNA only expressed very late in pollen development and highly up-regulated during the first 0.5 h of germination. The translation product of the S/D3 cDNA shares some features with a neuropeptide that regulates guidance and growth in the tips of extending axons. This study confirmed that the bulk of pollen mRNA accumulates well before germination, but that specific sequences are transcribed during the earliest moments of Fl-induced pollen germination.

Molecular and immunologic characterization of new isoforms of the Hevea brasiliensis latex allergen hev b 7: evidence of no cross-reactivity between hev b 7 isoforms and potato patatin and proteins from avocado and banana.

BACKGROUND: Hev b 7 is a Hevea brasiliensis latex allergen with sequence identities of 39% to 42% to patatins recently identified as potato allergens. The complementary DNAs encoding 2 different Hev b 7 isoforms were previously reported. OBJECTIVE: The aim of this study was to determine the sequence variation of Hev b 7 and to compare the IgE reactivity of individual isoforms in vitro and in vivo. A further objective was to evaluate possible cross-reactivities between Hev b 7 and patatins and proteins from banana and avocado. METHODS: An H brasiliensis lambda ZAP complementary DNA (cDNA) library was screened with use of a Hev b 7 cDNA probe. Four Hev b 7 isoforms were produced in recombinant form and their IgE-binding capacities were compared. IgE immunoblot inhibitions and ELISA inhibition assays were used to investigate the possible cross-reactivity between Hev b 7 and recombinant potato patatin and proteins from avocado and banana. RESULTS: Two new isoforms, S2 and D2, were identified by sequencing 32 cDNA clones with full-length coding regions. All 4 recombinant isoforms displayed esterase activity and identical IgE-binding capacities. The new isoforms S2 and D2 were evaluated in skin prick tests and provoked responses equivalent to natural Hev b 7. No cross-reactivity was observed between Hev b 7 isoforms and potato patatin and proteins from avocado and banana. CONCLUSIONS: All 4 recombinant Hev b 7 isoforms have equivalent IgE-binding capacity and therefore represent suitable reagents for the development of in vitro and in vivo diagnostic tests. Hev b 7, patatins, and their homologs appear not to contribute to cross-reactivity in the latex-fruit syndrome.

Toxicology of protein allergenicity: prediction and characterization.

The ability of exogenous proteins to cause respiratory and gastrointestinal allergy, and sometimes systemic anaphylactic reactions, is well known. What is not clear however, are the properties that confer on proteins the ability to induce allergic sensitization. With an expansion in the use of enzymes for industrial applications and consumer products, and a substantial and growing investment in the development of transgenic crop plants that express novel proteins introduced from other sources, the issue of protein allergenicity has assumed considerable toxicological significance. There is a need now for methods that will allow the accurate identification and characterization of potential protein allergens and for estimation of relative potency as a first step towards risk assessment. To address some of these issues, and to review progress that has been made in the toxicological investigation of respiratory and gastrointestinal allergy induced by proteins, a workshop, entitled the Toxicology of Protein Allergenicity: Prediction and Characterization, was convened at the 37th Annual Conference of the Society of Toxicology in Seattle, Washington (1998). The subject of protein allergenicity is considered here in the context of presentations made at that workshop.

Cloning and expression pattern of Hor v 9, the group 9 pollen isoallergen from barley.

In this study we report the cloning, sequence, and characterization of Hor v 9 allergen cDNAs from barley (Hordeum vulgare) pollen. Structural homologues of Kentucky bluegrass (Poa pratensis) group 9 pollen allergens were identified in a cDNA library of barley pollen expressed mRNAs. The Hor v 9 cDNA clone (hvp9742) contained an open reading frame encoding 313 amino acids which included a putative 27-residue signal peptide and one asparagine sequon for glycosylation. The mRNA corresponding to clone hvp9742 was produced abundantly in pollen during the late stages of anther development. The protein encoded by clone hvp974 was synthesized as a fusion protein in the E. coli expression vector pMAL. Immunoblots using antibodies to this recombinant allergen, rHor v 9, showed that Hor v 9 protein accumulated during pollen development and was produced maximally at pollen maturity. Using these antibodies, we also provide evidence that Hor v 9 protein localized to the extracellular matrix of mature pollen. Southern blots suggested that Hor v 9 allergens exist as multiple isoforms in barley. Sequence comparisons showed that the Hor v 9 cDNA clones were also homologous to group 5 allergens of Timothy grass (Phleum pratense) pollen and canary grass (Phalaris aquatica) pollen, and the group 9 allergen of ryegrass (Lolium perenne) pollen.

Stability of food allergens to digestion in vitro.

An integral part of the safety assessment of genetically modified plants is consideration of possible human health effects, especially food allergy. Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult because of the absence of valid methods and models. Food allergens may share physicochemical properties that distinguish them from nonallergens, properties that may be used as a tool to predict the inherent allergenicity of proteins newly introduced into the food supply by genetic engineering. One candidate property is stability to digestion. We have systematically evaluated the stability of food allergens that are active via the gastrointestinal tract in a simple model of gastric digestion, emphasizing the major allergens of plant-derived foods such as legumes (peanuts and soybean). Important food allergens were stable to digestion in the gastric model (simulated gastric fluid). For example, soybean beta-conglycinin was stable for 60 min. In contrast, nonallergenic food proteins, such as spinach ribulose bis-phosphate carboxylase/oxygenase, were digested in simulated gastric fluid within 15 sec. The data are consistent with the hypothesis that food allergens must exhibit sufficient gastric stability to reach the intestinal mucosa where absorption and sensitization (development of atopy) can occur. Thus, the stability to digestion is a significant and valid parameter that distinguishes food allergens from nonallergens.

Molecular characterization of Hor v 9. Conservation of a T-cell epitope among group IX pollen allergens and human VCAM and CD2.

We have cloned, sequenced and expressed a recombinant group IX pollen allergen from barley (Hordeum vulgare). Hor v 9 is a polypeptide of 313 amino acids. The Hor v 9 cDNA clone was engineered into the E. coli protein expression vector pMAL and expressed as a fusion of maltose binding protein and truncated Hor v 9. Polyclonal antibodies to the fusion protein were raised in mice. Cross-reactive proteins, RNA and DNA homologues were found in many agricultural species including wheat, rye, triticale, oats, maize, sunflower and flax. The presence of group IX-like proteins in a variety of agricultural crops may represent a previously uncharacterized aeroallergenic occupational hazard. Sequence comparisons of the barley allergen, Hor v 9, with Poa p 9 and other cloned group IX pollen allergens revealed putative structural domains common to all. These include a signal peptide, two conserved immunoglobulin-like motifs, a 150 amino acid highly conserved carboxyterminal domain and a carboxyterminal transmembrane helix. This structural arrangement is also found in cell adhesion molecules. The highly conserved T-cell epitope previously characterized and mapped in group IX allergens (and present in Hor v 9) was found in several human cell adhesion molecule sequences (VCAM, NCAM and CD2). This T-cell epitope corresponded to the most highly conserved amino acid residues common to all group IX homologues sequenced to date. CD2 and VCAM are known to play a role in allergic inflammation: VCAM is involved in the recruitment of lymphocytes to sites of inflammation, and cross-linking CD2 leads to T-cell activation. We anticipate that the similar structural arrangement of group IX allergens and human cell adhesion molecules, as well as the presence of a T-cell epitope common to group IX pollen allergens and cell adhesion molecules, will have important consequences in the natural history of the atopic immune response.

Assessment of the allergenic potential of foods derived from genetically engineered crop plants.

This article provides a science-based, decision tree approach to assess the allergenic concerns associated with the introduction of gene products into new plant varieties. The assessment focuses on the source from which the transferred gene was derived. Sources fall into three general categories: common allergenic food proteins; less common allergenic foods or other known allergen sources; and sources with no history of allergenicity. Information concerning the amino acid sequence identity to known allergenic proteins, in vitro and/or in vivo immunologic assays, and assessment of key physiochemical properties are included in reaching a recommendation on whether food derived from the genetically modified plant variety should be labeled as to the source of the transferred gene. In the end, a balanced judgement of all the available data generated during allergenicity assessment will assure the safety of foods derived from genetically engineered crops. Using the approaches described here, new plant varieties generated by genetic modification should be introduced into the marketplace with the same confidence that new plant varieties developed by traditional breeding have been introduced for decades.

Pollen allergen homologues in barley and other crop species.

Pollen from 10 agricultural plant species was surveyed for the presence of proteins crossreactive with group I, group IV and group IX allergens. Barley (Hordeum vulgare), maize (Zea mays), rye (Secale cerale), triticale (xTriticosecale cereale), oats (Avena sativa), Canola (Brassica napus) and sunflower (Helianthus annus) pollens contained numerous allergen cognate proteins. Northern blot analysis of barley pollen RNA revealed the presence of group I and group IX allergen transcripts. The barley pollen cDNA hvp9742, and three other cloned allergens: phlenum protense (Phl p) V, Phl p Va and Lolium perenne (Lol p) 1b, were demonstrated to have extensive nucleotide and amino acid sequence similarity to the Poa p IX isoallergens. It was concluded that hvp9742 represents a Poa p IX isoallergen homologue expressed by barley pollen, and was therefore designated Hor v IX. It is further shown that the most highly conserved domains of all seven proteins, including Hor v IX, map to previously defined Poa p IX antibody binding epitopes.