A three generation study with genetically modified Bt corn in rats: Biochemical and histopathological investigation.

For the last ten years, in accordance with the increased use of genetically modified (GM) foods for human and livestock, a large number of feeding studies have been carried out. However, the evidence is still far from proving whether the long-term consumption of GM foods poses a possible danger for human or animal health. Therefore, this study was designed to evaluate the effects of transgenic corn on the rats that were fed through three generations with either GM corn or its conventional counterpart. Tissue samples of stomach, duodenum, liver and kidney were obtained for histopathological examinations. The average diameter of glomeruli, thickness of renal cortex and glomerular volume were calculated and number of affected animals/number of examined animals for liver and kidney histopathology were determined. Amounts of urea, urea nitrogen, creatinine, uric acid, total protein, albumin and globulin were determined; enzyme activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyltransferase, creatine kinase and amylase were measured in serum samples. No statistically significant differences were found in relative organ weights of rats within groups but there were some minimal histopathological changes in liver and kidney. Changes in creatinine, total protein and globulin levels were also determined in biochemical analysis.     

Assessment of possible allergenicity of hypothetical ORFs in common food crops using current bioinformatic guidelines and its implications for the safety assessment of GM crops

Before a genetically modified (GM) crop can be commercialized it must pass through a rigorous regulatory process to verify that it is safe for human and animal consumption, and to the environment. One particular area of focus is the potential introduction of a known or cross-reactive allergen not previously present within the crop. The assessment of possible allergenicity uses the guidelines outlined by the Food and Agriculture Organization (FAO) and World Health Organization’s (WHO) Codex Alimentarius Commission (Codex) to evaluate all newly expressed proteins. Some regulatory authorities have broadened the scope of the assessment to include all DNA reading frames between stop codons across the insert and spanning the insert/genomic DNA junctions. To investigate the utility of this bioinformatic assessment, all naturally occurring stop-to-stop frames in the non-transgenic genomes of maize, rice, and soybean, as well as the human genome, were compared against the AllergenOnline (www.allergenonline.org) database using the Codex criteria. We discovered thousands of frames that exceeded the Codex defined threshold for potential cross-reactivity suggesting that evaluating hypothetical ORFs (stop-to-stop frames) has questionable value for making decisions on the safety of GM crops.     

A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats.

An animal model for safety assessment of genetically modified foods was tested as part of the SAFOTEST project. In a 90-day feeding study on Wistar rats, the transgenic KMD1 rice expressing Cry1Ab protein was compared to its non-transgenic parental wild type, Xiushui 11. The KMD1 rice contained 15mg Bt toxin/kg and based on the average feed consumption the daily intake was 0.54mg Bt toxin/kg body weight. No adverse effects on animal behaviour or weight gain were observed during the study. Blood samples collected one week prior to sacrifice were analyzed and compared for standard haematological and biochemical parameters. A few parameters were significantly different, but all within the normal reference intervals for rats of this breed and age and not in relation to any other findings, thus not considered treatment related. Upon sacrifice a large number of organs were weighed, macroscopic and histopathological examinations were performed with only minor changes to report. The aim of the study was to use a known animal model in performance of safety assessment of a GM crop, in this case KMD1 rice. The results show no adverse or toxic effects of KMD1 rice when tested in the design used in this 90-day study. Nevertheless the experiences from this study lead to the overall conclusion that safety assessment for unintended effects of a GM crop cannot be done without additional test group(s).     

A 90-day subchronic feeding study of genetically modified maize expressing Cry1Ac-M protein in Sprague–Dawley rats

The cry1Ac-M gene, coding one of Bacillus thuringiensis (Bt) crystal proteins, was introduced into maize H99 × Hi IIB genome to produce insect-resistant GM maize BT-38. The food safety assessment of the BT-38 maize was conducted in Sprague–Dawley rats by a 90-days feeding study. We incorporated maize grains from BT-38 and H99 × Hi IIB into rodent diets at three concentrations (12.5%, 25%, 50%) and administered to Sprague–Dawley rats (n = 10/sex/group) for 90 days. A commercialized rodent diet was fed to an additional group as control group. Body weight, feed consumption and toxicological response variables were measured, and gross as well as microscopic pathology were examined. Moreover, detection of residual Cry1Ac-M protein in the serum of rats fed with GM maize was conducted. No death or adverse effects were observed in the current feeding study. No adverse differences in the values of the response variables were observed between rats that consumed diets containing GM maize BT-38 and non-GM maize H99 × Hi IIB. No detectable Cry1Ac-M protein was found in the serum of rats after feeding diets containing GM maize for 3 months. The results demonstrated that BT-38 maize is as safe as conventional non-GM maize.     

Different Effects of Bacillus thuringiensis Toxin Cry1Ab on Midgut Cell Transmembrane Potential of Mythimna separata and Agrotis ipsilon Larvae

Bacillus thuringiensis (Bt) Cry toxins from the Cry1A family demonstrate significantly different toxicities against members of the family Noctuidae for unknown reasons. In this study, membrane potential was measured and analyzed in freshly isolated midgut samples from Mythimna separata and Agrotis ipsilon larvae under oral administration and in vitro incubation with Bt toxin Cry1Ab to elucidate the mechanism of action for further control of these pests. Bioassay results showed that the larvae of M. separata achieved a LD₅₀ of 258.84 ng/larva at 24 h after ingestion; M. separata larvae were at least eightfold more sensitive than A. ipsilon larvae to Cry1Ab. Force-feeding showed that the observed midgut apical-membrane potential (V_am) of M. separata larvae was significantly depolarized from −82.9 ± 6.6 mV to −19.9 ± 7.2 mV at 8 h after ingestion of 1 μg activated Cry1Ab, whereas no obvious changes were detected in A. ipsilon larvae with dosage of 5 μg Cry1Ab. The activated Cry1Ab caused a distinct concentration-dependent depolarization of the apical membrane; V_am was reduced by 50% after 14.7 ± 0.2, 9.8 ± 0.4, and 7.6 ± 0.6 min of treatment with 1, 5, and 10 μg/mL Cry1Ab, respectively. Cry1Ab showed a minimal effect on A. ipsilon larvae even at 20 μg/mL, and V_am decreased by 26.3% ± 2.3% after 15 min. The concentrations of Cry1Ab displayed no significant effect on the basolateral side of the epithelium. The V_am of A. ipsilon (−33.19 ± 6.29 mV, n = 51) was only half that of M. separata (−80.94 ± 6.95 mV, n = 75). The different degrees of sensitivity to Cry1Ab were speculatively associated with various habits, as well as the diverse physiological or biochemical characteristics of the midgut cell membranes.     

Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant—Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation

In addition to their applicability as biopesticides, Bacillus thuringiensis (Bt) Cry1Ac spore-crystals are being researched in the immunology field for their potential as adjuvants in mucosal and parenteral immunizations. We aimed to investigate the hematotoxicity and genotoxicity of Bt spore-crystals genetically modified to express Cry1Ac individually, administered orally (p.o.) or with a single intraperitoneal (i.p.) injection 24 h before euthanasia, to simulate the routes of mucosal and parenteral immunizations in Swiss mice. Blood samples were used to perform hemogram, and bone marrow was used for the micronucleus test. Cry1Ac presented cytotoxic effects on erythroid lineage in both routes, being more severe in the i.p. route, which also showed genotoxic effects. The greater severity noted in this route, mainly at 6.75 mg/kg, as well as the intermediate effects at 13.5 mg/kg, and the very low hematotoxicity at 27 mg/kg, suggested a possible inverse agonism. The higher immunogenicity for the p.o. route, particularly at 27 mg/kg, suggested that at this dose, Cry 1Ac could potentially be used as a mucosal adjuvant (but not in parenteral immunizations, due to the genotoxic effects observed). This potential should be investigated further, including making an evaluation of the proposed inverse agonism and carrying out cytokine profiling.     

Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control.

Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders--Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases.     

The application of post-market monitoring to novel foods.

The role of post-market monitoring (PMM) in the safety assessment of novel foods is critically discussed in order to derive guidelines as to in which situations the application of PMM might be warranted. Available data sources on food consumption and health status, and the methodologies for generating such data are reviewed. The paper suggests improvements to make them more applicable for PMM purposes. It is concluded that any PMM programme must be a hypothesis-driven scientific exercise. PMM can have a role as a complement to, but not as a replacement for, a comprehensive pre-market safety assessment. Its use may be appropriate to confirm that product use is as predicted in the pre-market assessment; to provide reassurance that effects observed in the pre-market assessment occur with no greater frequency or intensity in the post-market phase than anticipated; and to investigate the significance of any adverse effects reported by consumers after market-launch. However PMM is insufficiently powerful to test the hypothesis that any effects seen in the pre-market assessment are absent in the post-market phase. Current methodologies place limitations on what PMM can achieve. PMM should only be used when triggered by or when the focus is on specific evidence-based questions.     

Identification of Distinct Bacillus thuringiensis 4A4 Nematicidal Factors Using the Model Nematodes Pristionchus pacificus and Caenorhabditis elegans

Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using the model nematodes Pristionchus pacificus and Caenorhabditis elegans. We show that B. thuringiensis 4A4 kills both nematodes via intestinal damage. Whole genome sequencing of B. thuringiensis 4A4 identified Cry21Ha, Cry1Ba, Vip1/Vip2 and β-exotoxin as potential nematicidal factors. Only Cry21Ha showed toxicity to C. elegans, while neither Cry nor Vip toxins were active against P. pacificus, when expressed in E. coli. Purified crystals also failed to intoxicate P. pacificus, while autoclaved spore-crystal mixture of B. thuringiensis 4A4 retained toxicity, suggesting that primary β-exotoxin is responsible for P. pacificus killing. In support of this, we found that a β-exotoxin-deficient variant of B. thuringiensis 4A4, generated by plasmid curing lost virulence to the nematodes. Thus, using two model nematodes we revealed virulence factors of the nematicidal strain B. thuringiensis 4A4 and showed the multifactorial nature of its virulence.     

Assessing the risk of ballast water treatment to human health

Ballast water management systems utilizing noxious chemicals have to be approved according to the Ballast Water Convention by the International Maritime Organization (IMO). The approval procedure requires human health risk assessment. Our objective was to evaluate the existing human health risk assessment process for ballast water management systems. Towards this end, we analyzed the available applications for IMO approval. Since the majority of active substances currently in use are oxidative compounds the corresponding treatment systems generate and release a large number of disinfection by-products. The application dossiers only select a number of by-products for risk assessment. We propose a more comprehensive approach based on the type of ballast water management system, the quality of water treated and the toxicity of compounds discharged into the environment. Subsequent to effects assessment we propose to classify substances according to a hazard evaluation procedure, based on an approach used for maritime transport. We identified a need for better exposure assessment. This requires knowledge of exposure situations. We provide a comprehensive listing of occupational and non-occupational exposure settings and quantification models for exposure assessment.     

Toxicity of Parasporin-4 and Health Effects of Pro-parasporin-4 Diet in Mice

Parasporin-4 (PS4) is an aerolysin-type β-pore-forming toxin produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines; therefore, it is expected to be useful for the diagnosis and treatment of particular types of cancer cells. We examined the acute toxicity of PS4 on ICR mice. The LD₅₀ value was 160 μg/kg by a subcutaneous route. Potassium, ammonium, magnesium ion, creatinine, and urea nitrogen decreased in urine by the injection of PS4. Simultaneously, creatinine and urea nitrogen in mice serum increased. These results imply that PS4 impairs kidney function in mice. PS4 is obtained from Pro-parasporin-4 (ProPS4) by processing, and ProPS4 is produced by recombinant Escherichia coli as the inclusion body. The inclusion body of ProPS4 can be solubilized in a weak acid solution and activated by pepsin, implying that it would be solubilized and activated in the stomach of mammals after oral administration. Thus, the influence of the oral administration of it by C57BL/6J mice was examined. Although ProPS4 was activated to PS4 in the mouse digestive tract, any serious health hazard was not observed and there was no significant difference in body weight change.     

A model for probabilistic health impact assessment of exposure to food chemicals

A statistical model is presented extending the integrated probabilistic risk assessment (IPRA) model of van der Voet and Slob [van der Voet, H., Slob, W., 2007. Integration of probabilistic exposure assessment and probabilistic hazard characterisation. Risk Analysis, 27, 351–371]. The aim is to characterise the health impact due to one or more chemicals present in food causing one or more health effects. For chemicals with hardly any measurable safety problems we propose health impact characterisation by margins of exposure. In this probabilistic model not one margin of exposure is calculated, but rather a distribution of individual margins of exposure (IMoE) which allows quantifying the health impact for small parts of the population. A simple bar chart is proposed to represent the IMoE distribution and a lower bound (IMoEL) quantifies uncertainties in this distribution. It is described how IMoE distributions can be combined for dose-additive compounds and for different health effects. Health impact assessment critically depends on a subjective valuation of the health impact of a given health effect, and possibilities to implement this health impact valuation step are discussed. Examples show the possibilities of health impact characterisation and of integrating IMoE distributions. The paper also includes new proposals for modelling variable and uncertain factors describing food processing effects and intraspecies variation in sensitivity.     

Mode of action of mosquitocidal Bacillus thuringiensis toxins.

Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. Their primary action is to lyse midgut epithelial cells. In lepidopteran insects, Cry1A monomeric toxins interact with a first receptor and this interaction triggers toxin oligomerization. The oligomeric structure interacts then with a second GPI-anchored receptor that induces insertion into membrane microdomains and larvae death. In the case of mosquitocidal Bt strains, two different toxins participate, Cry and Cyt. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin-resistance. We will summarize recent findings on the identification of Cry receptors in mosquitoes and the mechanism of synergism: Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a Cry membrane-bound receptor.     

Effectiveness of the High Dose/Refuge Strategy for Managing Pest Resistance to Bacillus thuringiensis (Bt) Plants Expressing One or Two Toxins

To delay resistance development to Bacillus thuringiensis (Bt) plants expressing their own insecticide, the application of the Insect Resistance Management strategy called “High Dose/Refuge Strategy” (HD/R) is recommended by the US Environmental Protection Agency (US EPA). This strategy was developed for Bt plants expressing one toxin. Presently, however, new Bt plants that simultaneously express two toxins are on the market. We used a mathematical model to evaluate the efficiency of the HD/R strategy for both these Bt toxins. As the current two-toxin Bt plants do not express two new Cry toxins but reuse one toxin already in use with a one-toxin plant, we estimated the spread of resistance when the resistance alleles are not rare. This study assesses: (i) whether the two toxins have to be present in high concentration, and (ii) the impact of the relative size of the refuge zone on the evolution of resistance and population density. We concluded that for Bt plants expressing one toxin, a high concentration is an essential condition for resistance management. For the pyramided Bt plants, one toxin could be expressed at a low titer if the two toxins are used for the first time, and a small refuge zone is acceptable.     

High-throughput screening and small animal models,where are we?

Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limit its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the fish Danio rerio are gaining momentum as screening tools. These organisms combine genetic amenability, low cost and culture conditions that are compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a whole-animal context. Moreover, their use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. This review surveys the versatility of these animal models for drug discovery and discuss the options available at this day.     

Impact of food processing on the safety assessment for proteins introduced into biotechnology-derived soybean and corn crops

The food safety assessment of new agricultural crop varieties developed through biotechnology includes evaluation of the proteins introduced to impart desired traits. Safety assessments can include dietary risk assessments similar to those performed for chemicals intentionally, or inadvertently added to foods. For chemicals, it is assumed they are not degraded during processing of the crop into food fractions. For introduced proteins, the situation can be different. Proteins are highly dependent on physical forces in their environment to maintain appropriate three-dimensional structure that supports functional activity. Food crops such as corn and soy are not consumed raw but are extensively processed into various food fractions. During processing, proteins in corn and soy are subjected to harsh environmental conditions that drastically change the physical forces leading to denaturation and loss of protein function. These conditions include thermal processing, changes in pH, reducing agents, mechanical shearing etc. Studies have shown that processing of introduced proteins such as enzymes that impart herbicide tolerance or proteins that control insect pests leads to a complete loss of functional activity. Thus, dietary exposure to functionally active proteins in processed food products can be negligible and below levels of any safety concerns.