Safety assessment of Asterarcys quadricellulare, a microalga, with applications in poultry and livestock feed.

Asterarcys quadricellulare (AQ) is a microalgal species with potential applications in improving the quality of animal feed, and safety studies on this species are lacking. Therefore, this study presents safety data on an industrially cultivated strain of AQ tested using the following Organisation for Economic Co-operation and Development (OECD) guidelines: acute skin irritation in rabbits; skin sensitisation in guinea pigs; acute eye irritation in rabbits; acute oral fixed-dose procedure in rats; and bacterial reverse mutation using the B.N. Ames technique. Results showed that AQ is non-irritant and non-sensitising to skin. AQ caused transient conjunctival lacrimation and redness; however, the scores for these clinical signs translated into low ocular irritation indices and classification of AQ as non-irritant to the eyes. An acute oral dose of AQ (2000 mg/kg) did not cause mortality, change in body weight gain, or any general, functional, and neurobehavioral clinical signs. In five strains of Salmonella typhimurium bacteria, treatment with AQ did not cause biologically or statistically significant changes in the number of revertant colonies, indicating that AQ does not cause mutagenic toxicity. This study demonstrates the safety of a heterotrophically-produced strain of AQ and supports its use as a safe and non-toxic feed ingredient.

Toxicological evaluation of a glycan preparation from an enzymatic hydrolysis of Saccharomyces cerevisiae.

The aim of this paper was to provide a comprehensive toxicological and safety evaluation of a yeast cell wall preparation (YCWP) for use as an animal feed ingredient. The following toxicological assessments were carried out: the mutagenic activity was tested using the Ames' Test in five Salmonella typhimurium strains; clastogenic activity was investigated using the mammalian micronucleus test in Swiss ICO OF1 (IOPS Caw) mice; genotoxic activity was assessed using the in vitro mammalian chromosomal aberration test in human lymphocytes; acute oral toxicity was tested by administration of a single dose of 2000 mg/kg BW. Eye and skin irritation were assessed in rabbits according to OECD guidelines; skin sensitivity was established in guinea pigs by means of the Buehler test, while acute dermal and inhalation studies in rats were further completed, also according to OECD guidelines. All conducted tests were considered valid under the experimental conditions. No significant mutagenic activity or genotoxic activity was observed, and it was concluded that the test article did not induce any clastogenic effect. YCWP was found to be mildly irritating to the eye, slightly irritating to the skin but was found to be non-sensitizing in the guinea pig. The acute oral, dermal and inhalation studies did not yield any evidence of gross toxicity or pharmacological effects.

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

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

Dietary safety of a dual-enzyme preparation for animal feed: Acute and subchronic oral toxicity and genotoxicity studies.

Animal feed is routinely supplemented with exogenous enzymes to improve nutrient utilization, such as proteases to enhance protein hydrolysis in vivo and xylanases to alleviate feed related anti-nutritional factors. The present studies were conducted to evaluate the potential oral toxicity and genotoxicity of a dual-enzyme preparation, Vegpro® concentrate (VPr-C). Acute oral toxicity studies were conducted in adult male and female Sprague-Dawley Crl CD rats and CHS Swiss ICO:OFI (IOPS Caw) mice. Thirteen week preliminary and final subchronic oral toxicity studies were conducted in male and female rats. Genotoxicity was evaluated through a bacterial reverse mutation test (Ames test), an in-vitro mammalian chromosomal aberration test, and a mammalian micronucleus test. The LD50 was >2000 mg/kg of BW in mice and rats. In the 13-week oral toxicity study, the No Observed Adverse Effects Level (NOAEL) was 1000 mg/kg BW per day for females and 300 mg/kg BW per day for males. VPr-C showed no mutagenic activity in Salmonella typhimurium, did not induce significant chromosomal aberrations in cultured human lymphocytes, and did not increase the frequency or proportion of micronucleated immature erythrocytes in mice. There was no evidence of acute or subchronic toxicity or genotoxicity associated with the test article at these test dosages.

Patterns of gene expression in schistosomes: localization by whole mount in situ hybridization.

As a consequence of comprehensive transcriptome analysis followed by sequencing and draft assembly of the genome, the emphasis of schistosome research is shifting from the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mount in situ hybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes.

The polarity and magnitude of ambient charge influences three-dimensional neurite extension from DRGs.

Sulfated proteoglycans have inhibitory effects on neurite extension, and the negative charge of the glycosaminoglycan side chains may be involved in the inhibitory process. The main goal of this study is to investigate the effects of charge on three-dimensional neurite extension. Various concentrations of dermatan sulfate (DS), a chondroitin sulfate glycosaminoglycan, and consequently, various degrees of negative charge were presented on three-dimensional agarose hydrogels and the effect of charge on neurite extension from primary neurons was investigated. Dose-response experiments were also performed with the polycationic (positively charged) polysaccharide chitosan covalently coupled to agarose. The amount of DS or chitosan coupled to the agarose gel was quantified via metachromatic dye or Fourier transform infrared spectroscopy methods, respectively. The length of embryonic day 9 (E9) chick dorsal root ganglia neurites extended through charged agarose gels is dependent on the polarity and quantity of ambient charge. The inhibitory effects of the sulfated DS and the enhancing effects of the polycationic chitosan on neurite extension decrease as the amount of DS or chitosan coupled to agarose is decreased. These findings indicate that primary neural process extension is influenced by the polarity of ambient charge in a dose-responsive manner.

A laminin and nerve growth factor-laden three-dimensional scaffold for enhanced neurite extension.

Agarose hydrogel scaffolds were engineered to stimulate and guide neuronal process extension in three dimensions in vitro. The extracellular matrix (ECM) protein laminin (LN) was covalently coupled to agarose hydrogel using the bifunctional cross-linking reagent 1,19- carbonyldiimidazole (CDI). Compared to unmodified agarose gels, LN-modified agarose gels significantly enhanced neurite extension from three-dimensionally (3D) cultured embryonic day 9 (E9) chick dorsal root ganglia (DRGs), and PC 12 cells. After incubation of DRGs or PC 12 cells with YIGSR peptide or integrin beta1 antibody respectively, the neurite outgrowth promoting effects in LN-modified agarose gels were significantly decreased or abolished. These results indicate that DRG/PC 12 cell neurite outgrowth promoting effect of LN-modified agarose gels involves receptors for YIGSR/integrin beta1 subunits respectively. 1,2-bis(10, 12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC)-based lipid microcylinders were loaded with nerve growth factor (NGF), and embedded into agarose hydrogels. The resulting trophic factor gradients stimulated directional neurite extension from DRGs in agarose hydrogels. A PC 12 cell-based bioassay demonstrated that NGF-loaded lipid microcylinders can release physiologically relevant amounts of NGF for at least 7 days in vitro. Agarose hydrogel scaffolds may find application as biosynthetic 3D bridges that promote regeneration across severed nerve gaps.

The influence of physical structure and charge on neurite extension in a 3D hydrogel scaffold.

Understanding neural cell differentiation and neurite extension in three-dimensional scaffolds is critical for neural tissue engineering. This study explores the structure-function relationship between a 3D hydrogel scaffold and neural cell process extension and examines the role of ambient charge on neurite extension in 3D scaffolds. A range of agarose hydrogel concentrations was used to generate varied gel physical structures and the corresponding neurite extension was examined. Agarose gel concentration and the corresponding pore radius are important physical properties that influence neural cell function. The average pore radii of the gels were determined while the gel was in the hydrated state and in two different dehydrated states. As the gel concentration was increased, the average pore radius decreased exponentially. Similarly, the length of neurites extended by E9 chick DRGs cultured in agarose gels depends on gel concentration. The polycationic polysaccharide chitosan and the polyanionic polysaccharide alginate were used to incorporate charge into the 3D hydrogel scaffold, and neural cell response to charge was studied. Chitosan and alginate were covalently bound to the agarose hydrogel backbone using the bi-functional coupling agent 1,1'-carbonyldiimidazole. DRGs cultured in chitosan-coupled agarose gel exhibited a significant increase in neurite length compared to the unmodified agarose control. Conversely, the alginate-coupled agarose gels significantly inhibited neurite extension. This study demonstrates a strong, correlation between the ability of sensory ganglia to extend neurites in 3D gels and the hydrogel pore radius. In addition, our results demonstrate that charged biopolymers influence neurite extension in a polarity dependent manner.