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Pancreatic response of rats fed genetically modified soybean
Abstract
Mice fed genetically modified (GM) soybean were not affected in nutritional performance, but pancreatic microscopic features were disturbed. The mechanisms for these contradictory findings are unknown. This study analysed the histology of acinar pancreatic cells and the expression of pancreatitis-associated protein (PAP) and trypsinogen mRNA in rats fed GM soy protein. Two bioassays were run, each one with 34 Wistar rats distributed into two groups fed with non-GM or GM-soy protein (18% protein) for 0, 1, 3, 5, 15 and 30 days. Nutritional evaluation, plasma amylase levels, pancreatic histological analysis and quantification of PAP and trypsinogen mRNAs levels using quantitative real-time RT-PCR were done. No differences in nutritional performance among rats fed non-GM and GM diets were found. The GM, but not the non-GM, diet induced zymogen-granule depletion after 15 days feeding, returning to normal levels after 30 days (P < 0.05). Acinar disorganization started as early as 5 days after initiation of the GM diet and it recovered after 30 days. Levels of PAP mRNA significantly increased in the GM diet between day 1 and day 3 and decreased to the basal level by day 15. Trypsinogen mRNA peaked at two different times; at day 1 and at day 15, decreasing to basal levels after 30 days. Plasma amylase levels remained unchanged at all times. This indicates that GM soy protein intake affected pancreas function, evidenced by the early acute PAP mRNA increased levels and pancreas cellular changes followed by recuperation of acinar cells after 30 days.
... Consequently, the studies by the Malatesta's group (Malatesta et al., 2005(Malatesta et al., , 2008bCisterna et al., 2008) at the microscopic and ultramicroscopic levels showed cellular changes attributable to GM soybean intake. Magaña-Gómez et al. (2008) conducted a study in Wistar rats, in which the hypothesis was that the intake of GM (SUPRO 500E) soybean could induce pancreatic stress or injury by analyzing the expression of pancreatitis-associated protein (PAP) and trypsinogens by qRT-PCR in rats fed GM soy protein for 30 days. The hypothesis was based on the results of previous investigations showing that mice chronically fed since gestation with GM had problems in synthesis and processing of zymogens by pancreatic acinar cells and reduced nucleoplasmic and nucleolar and perichromatin granule accumulation on pancreatic acinar cell nuclei (Malatesta et al., 2002b(Malatesta et al., , 2003. ...
... The hypothesis was based on the results of previous investigations showing that mice chronically fed since gestation with GM had problems in synthesis and processing of zymogens by pancreatic acinar cells and reduced nucleoplasmic and nucleolar and perichromatin granule accumulation on pancreatic acinar cell nuclei (Malatesta et al., 2002b(Malatesta et al., , 2003. Magaña-Gómez et al. (2008) did not find differences in nutritional performance among rats fed non-GM and GM diets. The GM diet induced significant zymogen-granule depletion after 15 days feeding, returning to normal levels after 30 days. ...
... Similarly, scientific controversy is also present in relation to the safety of GM soybeans. While it has been reported that 356043 (Sakamoto et al., 2007) and 305423 soybeans were as safe as conventional non-GM soybeans, some authors are still concerned by the safety of GM soybeans and recommend to investigate the long-term consequences of GM diets and the potential synergistic effects with other products and/or conditions (Malatesta et al., 2008a,b;Cisterna et al., 2008;Magaña-Gómez et al., 2008). ...
In recent years, there has been a notable concern on the safety of genetically modified (GM) foods/plants, an important and complex area of research, which demands rigorous standards. Diverse groups including consumers and environmental Non Governmental Organizations (NGO) have suggested that all GM foods/plants should be subjected to long-term animal feeding studies before approval for human consumption. In 2000 and 2006, we reviewed the information published in international scientific journals, noting that the number of references concerning human and animal toxicological/health risks studies on GM foods/plants was very limited. The main goal of the present review was to assess the current state-of-the-art regarding the potential adverse effects/safety assessment of GM plants for human consumption. The number of citations found in databases (PubMed and Scopus) has dramatically increased since 2006. However, new information on products such as potatoes, cucumber, peas or tomatoes, among others was not available. Corn/maize, rice, and soybeans were included in the present review. An equilibrium in the number research groups suggesting, on the basis of their studies, that a number of varieties of GM products (mainly maize and soybeans) are as safe and nutritious as the respective conventional non-GM plant, and those raising still serious concerns, was currently observed. Nevertheless, it should be noted that most of these studies have been conducted by biotechnology companies responsible of commercializing these GM plants. These findings suggest a notable advance in comparison with the lack of studies published in recent years in scientific journals by those companies. All this recent information is herein critically reviewed.
... The authors concluded that GTS protein intake affected pancreatic function, as evidenced by the increased levels of early acute PAP mRNA and cellular changes in the pancreas, followed by regeneration at 15 days and full recuperation of acinar cells after 30 days. 57 GTS was also analyzed for metabolic effects in rabbits. 58 The animals received a diet containing 20% soybean meal from GTS or conventional soybeans (representing around 65% of the total protein requirements for rabbits 58 ) for 40 Ϯ 5 days. ...
... In publications reviewed by Pryme and Lembcke, 42 as well as in those mentioned in this review, the conclusions have varied from no alteration of the nutritional value of the GM food tested, 11,45,51,61,65,67,71,80 to minimal detrimental effects on the nutritional value, 81 to in vivo submicroscopic effects in different animal species. 23,54,55,57,82 Animal models used to test GM foods have been diverse, including rats, mice, cattle, fish, and poultry, and the assay periods have also varied. Some of the studies did not use microscopic, biochemical, or in vivo indicators to test for effects in the animals. ...
... 51,65 The most common result has been that there were no effects at the macroscopic level; however, organelles and other subcellular structures are clearly affected, as shown at ultramicroscopic levels. [52][53][54][55][56][57][58]59,68,73,74,76 The necessity of testing GM crops case by case has been established. Therefore, efforts should be directed towards finding the best experimental design, taking into consideration the inclusion levels of GM food and the appropriate animal model to detect effects that can occur in a human organism. ...
The risk assessment of genetically modified (GM) crops for human nutrition and health has not been systematic. Evaluations for each GM crop or trait have been conducted using different feeding periods, animal models, and parameters. The most common result is that GM and conventional sources induce similar nutritional performance and growth in animals. However, adverse microscopic and molecular effects of some GM foods in different organs or tissues have been reported. Diversity among the methods and results of the risk assessments reflects the complexity of the subject. While there are currently no standardized methods to evaluate the safety of GM foods, attempts towards harmonization are on the way. More scientific effort is necessary in order to build confidence in the evaluation and acceptance of GM foods.
... Por su condición de apéndice del MGC, el MIG aplicado al maíz, suma al desconocimiento a priori de los efectos epistáticos, pleiotrópicos y colaterales del MGC, el desconocimiento a priori derivado de la transformación en sí, ya que el MIG usa el producto del MGC como base para la transformación. Hay evidencias a posteriori que los efectos adicionales derivados de la transformación a pesar de su proclamada superior precisión, podrían no ser inocuos al consumidor, como se deriva de estudios con animales de laboratorio (Serallini et al., 2007;Magaña-Gómez et al., 2008). ...
... Una evaluación de esos efectos requeriría el diseño factorial 2 2 , que incluyera a un híbrido comercial no transgénico de uso comercial en el agro-ecosistema, al híbrido de isolíneas, y las versiones transgénicas de ambos. superior accuracy; they may not be safe for consumers, as known by studies with laboratory animals (Serallini et al., 2007;Magaña-Gómez et al., 2008). ...
This essay discusses two statements: a) there are no fundamental differences between classical genetic improvement (CGI) or breeding and genetic engineering improvement (GEI); the latter is an extension of the first but is more accurate so, it gives greater safety to the consumer; and b) transformation by genetic engineering is equivalent to a natural mutation. CGI and the GEI pursue objectives that can be complementary; but its foundations, methods and biological implications are different: the CGI works within the limits of sexual compatibility, using the species genetic diversity as a favorable source of DNA, which gives the precision and recombination mechanisms of sexual reproduction. The CGI uses foreign DNA integrated into a transgenic chimera, which is inserted imprecisely known in the chromosomal space of the transforming and generates a different locus in each independent transgenic event. Such dispersion can lead to an accumulation of transgenic chimeras in their progeny, and possible deleterious effects. By excluding the quantitative phenotypic characteristics as yield, GEI depends on the progress made by the CGI to develop superior phenotypes. It's also concurred with the statement that, the transgenic insertion is a mutation with different implications for maize genome. The expression of each allele is regulated by the genome and epigenome to be activated or deactivated according to a development plan for growth of the genotype. In contrast, the transgene mutant is expressed somatically (in all plant cells, without pause).
... Por su condición de apéndice del MGC, el MIG aplicado al maíz, suma al desconocimiento a priori de los efectos epistáticos, pleiotrópicos y colaterales del MGC, el desconocimiento a priori derivado de la transformación en sí, ya que el MIG usa el producto del MGC como base para la transformación. Hay evidencias a posteriori que los efectos adicionales derivados de la transformación a pesar de su proclamada superior precisión, podrían no ser inocuos al consumidor, como se deriva de estudios con animales de laboratorio (Serallini et al., 2007;Magaña-Gómez et al., 2008). ...
... Una evaluación de esos efectos requeriría el diseño factorial 2 2 , que incluyera a un híbrido comercial no transgénico de uso comercial en el agro-ecosistema, al híbrido de isolíneas, y las versiones transgénicas de ambos. superior accuracy; they may not be safe for consumers, as known by studies with laboratory animals (Serallini et al., 2007;Magaña-Gómez et al., 2008). ...
This essay discusses two statements: a) there are no fundamental differences between classical genetic improvement (CGI) or breeding and genetic engineering improvement (GEI); the latter is an extension of the first but is more accurate so, it gives greater safety to the consumer; and b) transformation by genetic engineering is equivalent to a natural mutation. CGI and the GEI pursue objectives that can be complementary; but its foundations, methods and biological implications are different: the CGI works within the limits of sexual compatibility, using the species genetic diversity as a favorable source of DNA, which gives the precision and recombination mechanisms of sexual reproduction. The CGI uses foreign DNA integrated into a transgenic chimera, which is inserted imprecisely known in the chromosomal space of the transforming and generates a different locus in each independent transgenic event. Such dispersion can lead to an accumulation of transgenic chimeras in their progeny, and possible deleterious effects. By excluding the quantitative phenotypic characteristics as yield, GEI depends on the progress made by the CGI to develop superior phenotypes. It¿s also concurred with the statement that, the transgenic insertion is a mutation with different implications for maize genome. The expression of each allele is regulated by the genome and epigenome to be activated or deactivated according to a development plan for growth of the genotype. In contrast, the transgene mutant is expressed somatically (in all plant cells, without pause).
... In this study, although no significant differences of AST and ALT was observed among transgenic soybean DBN9004, DBN8002 groups and JACK group, the higher level of AST and ALT in liver of DBN9004 and DBN8002 groups indicated that the function of hepatocytes may be influenced by the bacterial toxins or be related to sampling error. In the present study, no significant difference in enzymatic activities was found in the head kidney, liver and spleen of channel catfish fed with transgenic soybeans and JACK, consistent to the findings by Gao et al. and Magaña-Gómez et al., which documented that the plasma amylase level of mice fed with transgenic soybean and the activities of SOD, malondialdehyde (MDA) and CAT of zebrafish larvae exposed to two Bt proteins were unaltered, respectively 40,41 . Moreover, a similar finding was reported by Sanden et al. that no significant difference of AST and ALT in plasma of Atlantic salmon fed GM corn and conventional soybean for eight months 42 . ...
Transgenic soybean is one of the most planted crops for human food and animal feed. The channel catfish (Ictalurus punctatus) is an important aquatic organism cultured worldwide. In this study, the effect of six different soybean diets containing: two transgenic soybeans expressing different types of cp4-epsps, Vip3Aa and pat genes (DBN9004 and DBN8002), their non-transgenic parent JACK, and three conventional soybean varieties (Dongsheng3, Dongsheng7, and Dongsheng9) was investigated in juvenile channel catfish for eight weeks, and a safety assessment was performed. During the experiment, no difference in survival rate was observed in six groups. The hepatosomatic index (HSI) and condition factor (CF) showed no significant difference. Moreover, comparable feed conversion (FC), feeding rate (FR), and feed conversion ratio (FCR) were found between transgenic soybean and JACK groups. Assessment of growth performance showed that the weight gain rate (WGR) and specific growth rate (SGR) of channel catfish were consistent. In addition, there were no changes in enzyme activity indexes (lactate dehydrogenase (LDH), total antioxidant capacity (T-AOC), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) in channel catfish among treatments. The research provided an experimental basis for the aquaculture feed industry to employ transgenic soybean DBN9004 and DBN8002 for commercial purposes.
... Neither pathological there have been signs or documented anomalies. It has been concluded the transgenic rice, including T-cell oral administration Japanese cedar epitopes, did not have any adverse effects every day they were free to feed [37,38,39,40,41,42,43,44,45]. ...
The health of genetically engineered foods/plants, which is one of the significant issues has been raised in recent years. Various non-governmental organizations and customers recommended that all GM foods before authorization for human consumption should be subject to long-term animal feed studies. The fundamental purpose of this review is to assess the new potential harmful impact/safety assessment of genetically engineered plants for the use of humans. A balance in the number of research groups, depending on their research, a variety of GM crops (maize and soybeans in particular) are varied as for traditional non-genetically modified plants. It is worth remembering that most of the experiments were carried out in biotechnology firms that sell these GM plants. In this review, we discussed in detail the risk assessment of genetically modified plants.
... Reports commissioned by biotechnology companies and their associates report that genetically modified (GM) produce such as GM maize and soybeans are as safe as naturally occurring plants or crops (Domingo and Bordonaba 2011;Domingo 2016). However, similar studies by independent groups conclude otherwise (Séralini et al. 2011;Magaña-Gómez et al. 2008;de Vendômois et al. 2009). Humans continue to have limited knowledge and information about the carcinogenicity, teratogenicity, and mutagenicity of GM foods (Domingo and Bordonaba 2011). ...
The Malaysian government recognises the potential contribution of biotechnology to the national economy. However, ongoing controversy persists regarding its ethical status and no specific ethical guidelines have been published relating to its use. In developing such guidelines, it is important to identify the underlying principles that are acceptable to Malaysian society. This paper discusses the process of determining relevant secular and Islamic ethical principles and establishing their similarities before harmonising them. To achieve this, a series of focus group discussions were conducted with 23 knowledge experts representing various stakeholders in the biotechnology community. Notably, several principles between the secular and Islamic perspectives are indirectly or directly similar. All the experts agreed with the predominant six ethical principles of secular and Islamic philosophy and their importance and relevance in modern biotechnology. These are beneficence and non-maleficence as the main or overarching principles, the preservation of religious and moral values, the preservation of the intellect and the mind, the protection of human safety, the protection of future generations, and protection of the environment and biological diversity. Several adjustments were made to the terminologies and definitions of these six principles to formulate acceptable guiding principles for the ethics of modern biotechnology in Malaysia. These can then be adopted as core values to underpin future national guidelines on modern biotechnology ethics. These principles will be particularly important in guiding the policy makers, enforcers, industries and researchers to streamline their activities. In so doing, modern biotechnology and its products can be properly managed without jeopardising the interests of the Muslim community as well as the general public. Importantly, they are expansive and inclusive enough to embrace the religious sensitivity of diverse quarters of Malaysia.
... Therefore, it would be almost impossible to analyse the intake effects of a new GM food as a whole with a single experimental model (EFSA 2008;Malatesta 2009). For instance, different experimental models that were used to evaluate the intake effect of the same GM food yielded highly variable conclusions, from no adverse effects to negative ones (Brake and Evenson 2004;Gu et al. 2013;Magaña-Gomez et al. 2008;Malatesta et al. 2002). Although data analysis could be different, the measured biological or biochemi cal indicators were not the same either, further demonstrating the complexity of these evaluation tasks. ...
Food products from genetically modified (GM) crops for human consumption, referred to as ‘GM foods’ in this chapter, have been available on the market since 1994. A few years after their introduction, controversy and negative reactions arose because of their potentially harmful environmental effects and the health risks associated with their intake. In spite of ongoing concerns, the growing of GM crops has increased more than 100-fold during the last 20 years. Nowadays, one of the most controversial aspects of GM foods consumption is still the risk to human health. The first complaint is that genes are inserted randomly into the crop genome that in turn may modify non-target gene sequences or cause genetic mutations during the transformation process. Therefore, the insertion site and copies of the new DNA sequence should be analysed before the approval of the product for human consumption. This raises the possibility of new conjugated proteins or peptides with unknown biological effects on human health. Another critical point to consider is the presence of proteins without a natural history of consumption. Recently, the potential carcinogenicity of herbicides used in GM crop land has been used to argue for the lack of safety of GM foods. The majority of results show that GM foods have no adverse effects on model animals. Only a few studies have detected serious risks attributable to some GM foods, principally at the microscopic and molecular levels. In this respect, assessment of the health risk associated with GM foods has not been standardized and, as a result, a wide variety of protocols are being used. In the near future, it will be possible to study the effects of specific nutrients, foods or whole diets on the expression of thousands of genes at the same time, as well as changes in metabolic pathways or the use of specific metabolites as biomarkers for human intake or animal models. This ‘omics’ approach will be an alternative for characterizing the health risks of any GM food. This chapter reviews the current published information on nutrition and health concerns associated with the consumption of GM foods, presents the pros and cons for GM food consumption, and discusses some future perspectives on the risk assessment based on current science and technology developments.
... We have also witnessed scientific controversy over the use and safety of transgenic soybean. Soybean 356043 (Sakamoto et al. 2007) and soybean 305423 have been reported as safe as conventional non-transgenic soybeans, but raise concern and recommend to evaluate the long-term consequences of GM diets and the potential synergistic effects with other products and/or conditions (Cisterna et al. 2008;Magaña-Gómez et al. 2008;Malatesta et al. 2008). Ladics et al. (2003) reported that approximately 2% of adult and nearly 5% of children are prone to food allergy. ...
... We have also witnessed scientific controversy over the use and safety of transgenic soybean. Soybean 356043 (Sakamoto et al. 2007) and soybean 305423 have been reported as safe as conventional non-transgenic soybeans, but raise concern and recommend to evaluate the long-term consequences of GM diets and the potential synergistic effects with other products and/or conditions (Cisterna et al. 2008;Magaña-Gómez et al. 2008;Malatesta et al. 2008). Ladics et al. (2003) reported that approximately 2% of adult and nearly 5% of children are prone to food allergy. ...
... This suggests that the statistical methods used in the analyses were inappropriate (i.e., the highly dependent nature of the observed measures makes it unlikely that any of the findings reported would have reached statistical significance). In (Magana-Gomez et al., 2008) the standard errors reported for body weight gains and food consumption rates were exactly the same for both groups (Table 4). Standard errors for feed conversion rates and protein efficiency measures were also the same for both groups. ...
IntroductionThe General Strategic Situation of the Debate About Green Biotechnology TodayThe aim of this text is to set the framework for a better communication about science and regulation, and production of GM crops. GM stands for Genetic Modification, basically an unfortunate denomination, because actually all crops are genetically modified, but it is a worldwide accepted term for genetically engineered crops, including transgenes, auto- and allotransgenes, cis- and infra-genes, and synthetic genes, for details see Beardmore [1]. By including gene stacking of various kinds, the situation is getting even more complex [2]. With the introduction of in Vivo Mutation (with Zink-Finger Technology and the latest transformation method transcription activator-like family of type III effectors [TALEs]) the situation will change even more, the age ...
... Preparation and mixing of foods potentially enhance or impede nutrient bioavailability, taboos regulate intake, and dietary acculturation after migration alters nutritional status (Freimer et al., 1983;Mintz and DuBois, 2002). Agricultural practices, erosion, and leaching have depleted soils of nutrients in some areas (see review by Khoshgoftarmanesh et al., 2010), while potential negative effects of genetic modification of foods coupled with lack of complete knowledge concerning bioactive elements and pesticides create pressures in others (Cellini et al., 2004;Domingo, 2007;Kulper et al., 2001;Magana-Gomez et al., 2008;Walters, 2004). All of these factors carry the potential to become selective pressures when introduced to newly arrived individuals. ...
... A study on mice, into whose diet hemagglutinins was introduced, demonstrated that lectins can induce structural and enzyme liver changes [17]. Another study investigated the mechanism of action of these lectins and proposed the hypothesis of two mechanisms: interfering with cellular repair mechanisms for affected structures and blocking of mucus secretion [18]. ...
Diet can influence the structural characteristics of internal organs. An experiment involving 130 meat broilers was conducted during 42 days (life term for a meat broiler) to study the effect of feed with protein from genetically modified soy. The 1-day-old birds were randomly allocated to five study groups, fed with soy, sunflower, wheat, fish flour, PC starter. In the diet of each group, an amount of protein from soy was replaced with genetically modified soy (I - 0%, II - 25%, III - 50%, IV - 75%, V - 100% protein from genetically modified soy). The level of protein in soy, either modified, or non-modified, was the same. Organs and carcass weights were measured at about 42 days of age of the birds and histopathology exams were performed during May-June 2009. No statistically significant differences were observed in mortality, growth performance variables or carcass and organ yields between broilers consuming diets produced with genetically modified soybean fractions and those consuming diets produced with near-isoline control soybean fractions. Inflammatory and degenerative liver lesions, muscle hypertrophy, hemorrhagic necrosis of bursa, kidney focal tubular necrosis, necrosis and superficial ulceration of bowel and pancreatic dystrophies were found in tissues from broilers fed on protein from genetically modified soy. Different types of lesions found in our study might be due to other causes (parasites, viral) superimposed but their presence exclusively in groups fed with modified soy raises some serious questions about the consequences of use of this type of feed.
... "The results appear to indicate that rats fed on a GM diet had a pancreatic supraphysiological stimuli or synergism with cholecystokinin (CCK); although not severe, it was sufficiently strong to induce a mild pancreatic injury with an adaptive response" (p. 224 Magaña-Gómez et al., 2008). ...
The Commerce Commission requested that I research and report to the Commission on
whether animals exposed to feed containing genetically modified material (“GM feed”)
do in fact contain “no GM [genetically modified] ingredients”. The provision of expert
opinion to the Commission was sought in relation to ‘Inghams Enterprises (NZ) Pty
Limited chicken product/s as advertised as containing “no added hormones, GM
[genetically modified] ingredients” and sold in New Zealand. I was to comment on
(including comment on the likelihood of the event occurring) with regard to GM plants
used in food or feed:
• could DNA from GM plants be transferred to the animal;
• could GM plants be incorporated into other products sold as chicken products,
including breading or stuffing;
• could proteins from GM plants be transferred to the product or could the GM feed
alter metabolites in the animal;
• could the GM feed cause physiological or immunological responses in the
animal?
GM crops are the most studied crops in history. Approximately 5% of the safety studies on them show adverse effects that are a cause for concern, and tend to be featured in media reports. Although these reports are based on just a handful of GM events, they are used to cast doubt on all GM crops. Furthermore, they tend to come from just a few laboratories and are published in less important journals. Importantly, a close examination of these reports invariably shows methodological flaws that invalidate any conclusions of adverse effects. Twenty years after commercial cultivation of GM crops began, a bona fide report of an adverse health effect due to a commercialized modification in a crop has yet to be reported. This article is protected by copyright. All rights reserved.
Genomic technologies started in the early 1980s to improve the genomes of cultivated crop species. For example the term “Bt” comes from the soil bacterium Bacillus thuringiensis containing genes, e.g. Cry1Ac, Cry2Ab, Cry1F, Cry3Bb1, that provides protection against lepidopteran insect pests. Those genes have been inserted in crops such as corn, cotton, soybean, rice, potato and canola released for cultivation in mid 1990s in USA, and later in many other countries like China and India. About 29 countries commercialized genetically-modified (GM) or ‘transgenic’ crops while 30 countries granted regulatory approvals for planting GM-crops; together making 75 % of the world population. Potential harmful effects of the Bt-crops on non-targets were quantified before releasing such non-conventional crops into the environment. The cultivation of Bt-crops were most commonly found safe, based on various studies including the insertional impact of transgene and its regulatory elements on plant phenotype and agronomic performance, effect on non-target organisms (NTOs) and nutritional impacts on multiple experimental models. Albeit the studies were conducted for limited durations. However, the skeptics always claim for conducting extensive clinical as well as field trials, and also doubt on methods and procedures of calculating the ecological risks. This debate is still on-going, especially after reports on substantial reduction of monarch butterfly caterpillars exposed to Bt-maize pollen, though later nullified; and detection of traces of transgene in various tissues of experimental animals. Procedures, methods and protocols for evaluating potential risks of GM-crops and foods should be standardized as the first step to build trust of researchers and end-users. Many efforts should be exerted in deploying genes of interest, marker genes and regulatory sequences invoking no or little issues of potential risks to the ecosystem.
Introduction: Genotypes establish the developmental parameters and constraints on human traits, but environmental factors interacting with genes ultimately shape many phenotypes. These resulting phenotypes must prove beneficial for both survival and reproduction to continue any species. During their evolutionary history, human beings have demonstrated great capacity for survival and reproductive success while adapting to a wide range of environments. By interacting with increasingly complex cultural strategies to manage environmental advantages, genetic variation found in the human genome supported the human ancestral migrations out of Africa and around the globe. However, the manner by which genes and environment communicate to affect phenotypes remains unclear. The plasticity seen in fetal development and neonatal growth patterns appears to reflect the presence of early physiological acclimatization to environmental signals (Cutfield et al., 2007). Such signals leave lasting directives for gene expression, metabolic pathways and future adaptation capabilities without DNA mutation (Waterland and Garza, 1999). Additionally, these signals provide potentially reversible regulations of gene expression that more rapidly respond to environmental fluctuation than responses originating through changes in the DNA sequencing (Burdge et al., 2007). These “epigenetic” regulatory signals are transgenerational (Haig, 2004), yet the patterns are less robust across generations given their responsiveness to the environment. The reversible nature of these signal mechanisms leaves many unanswered questions regarding the mode of environmental influence on phenotypic development during human evolution. Today’s migrating populations experiencing rapid and dramatic changes in climate, food chain, social, and economic factors provide a unique model for testing the epigenetic hypotheses in humans.
Öz: Üretimi gittikçe artan ve toksikolojik yönden incelenmeden sofralarımıza daha sık giren GDO’lu ürünlerin sağlığımıza ne gibi etkileri olacağı da belirsizdir. Alerjik tepkimelere yol açabileceği, besin zinciri içinde birikebileceği, toksik etkiler yapabileceği ve antibiyotik direnci oluşturabileceği gibi etkiler bunların arasında en önemlileridir. Toksikolojik çalışmalar GD ürün üzerinde 90-günlük, uzun dönem ve çoklu jenerasyon olarak yapılmıştır. En çok çalışma yapılan ürünlerin başında mısır, soya fasulyesi ve pirinç gelmektedir. Çalışmalarda OECD Guideline’da (1998) belirtilen veya EFSA (2008) tarafından tartışılan genel prensipler GDO içeren diyetle beslemenin güvenliği için uygulanmış veya duruma göre modifiye edilerek düzenlenmiştir. Bu derlemede GDO’ların kullanım alanları, avantajları ve dezavantajları, toksikolojik etkileri üzerine yapılmış çalışmalar ve Türkiye’deki yasal mevzuat incelenmiştir.
Summary : The impact of GMO on human and animal health is still an uncertain debate; where these products are consumed without completing the toxicity researches. The adverse health effects include allergic reactions, toxic reactions and antibiotic resistance while these compounds accumulate in food chain. These researches on GMO and related products generally include the 90-day, long period and multiple generation toxicity studies; where corn soybean and rice were the most studied ones. General principles mentioned in OECD Guideline (1998) or discussed by EFSA (2008) were applied or regulated by modifying according to condition for safety of the diet including GMO. In this review, the uses of GMOs, advantages and disadvantages, related toxicity studies and legal regulations in Turkey were discussed.
There is disagreement internationally across major regulatory jurisdictions on the relevance and utility of whole food (WF) toxicity studies on GM crops, with no harmonization of data or regulatory requirements. The scientific value, and therefore animal ethics, of WF studies on GM crops is a matter addressable from the wealth of data available on commercialized GM crops and WF studies on irradiated foods. We reviewed available GM crop WF studies and considered the extent to which they add to the information from agronomic and compositional analyses. No WF toxicity study was identified that convincingly demonstrated toxicological concern or that called into question the adequacy, sufficiency, and reliability of safety assessments based on crop molecular characterization, transgene source, agronomic characteristics, and/or compositional analysis of the GM crop and its near-isogenic line. Predictions of safety based on crop genetics and compositional analyses have provided complete concordance with the results of well-conducted animal testing. However, this concordance is primarily due to the improbability of de novo generation of toxic substances in crop plants using genetic engineering practices and due to the weakness of WF toxicity studies in general. Thus, based on the comparative robustness and reliability of compositional and agronomic considerations and on the absence of any scientific basis for a significant potential for de novo generation of toxicologically significant compositional alterations as a sole result of transgene insertion, the conclusion of this review is that WF animal toxicity studies are unnecessary and scientifically unjustifiable.
Abstract To determine the reliability of food safety studies carried out in rodents with genetically modified (GM) crops, a Food Safety Study Reliability Tool (FSSRTool) was adapted from the European Centre for the Validation of Alternative Methods' ToxRTool. Reliability was defined as the inherent quality of the study with regard to use of standardized testing methodology, full documentation of experimental procedures and results, and the plausibility of the findings. Codex guidelines for GM crop safety evaluations indicate toxicology studies are not needed when comparability of the GM crop to its conventional counterpart has been demonstrated. This guidance notwithstanding, animal feeding studies have routinely been conducted with GM crops, but their conclusions on safety are not always consistent. To accurately evaluate potential risks from GM crops, risk assessors need clearly interpretable results from reliable studies. The development of the FSSRTool, which provides the user with a means of assessing the reliability of a toxicology study to inform risk assessment, is discussed. Its application to the body of literature on GM crop food safety studies demonstrates that reliable studies report no toxicologically relevant differences between rodents fed GM crops or their non-GM comparators.
The safety assessment of genetically modified (GM) plants and their derived food and feed is nowadays a matter of lively debate. Any nutritional consequence of GM food consumption needs to be considered, both in terms of possible changes in the levels of nutrients in the food itself, and in terms of direct or indirect, immediate or cumulative effects on the overall diet. From this point of view, several potential risks have been taken into consideration by scientists when evaluating the safety of GM organisms (GMOs) by animal feeding trials; in particular, the possibility has been considered that GM food effects might not be due to the introduced gene itself, but to the unintended consequences of its introduction, such as allergy induction, DNA transfer to gastrointestinal flora (including antibiotic resistance), production of toxic compounds (i.e. insecticidal proteins), persistence of herbicide residues in transgenic herbicide-tolerant plants, any kind of unintended effects associated with the expression of recombinant proteins in non-native hosts. In this review, an overview of the literature data is reported on the effects of GMO intake in different animal models, with the scope to outline the state of the art of the scientific research on this subject.
The presence of DNA fragments in tissues from rabbits given genetically modified (GM) soya-bean meal (solvent extracted) was investigated by using the polymerase chain reaction (PCR) approach. Moreover, the possible effects on cell metabolism were evaluated by determination of several specific enzymes in serum, heart, skeletal muscle, liver and kidney. The chloroplast sequence for tRNA Leu by using the Clor1/Clor2 primers designed on chloroplast trnL sequence was clearly detected. On the contrary, two couples of species specific primers for conventional (Le1-5/Le 1-3 which amplifies the soya bean lectin gene) and genetically modified (35S1/35S2 which amplifies the 35S CMV promoter that is present in the genomic structure of GM soya bean) soya bean were not found in all samples. No differences in enzyme levels were detected in serum, but a significant increase of lactic dehydrogenase, mainly concerning the LDH1 isoenzyme was found in particular in kidney and heart but not in the muscle, thus suggesting a potential alteration in the local production of the enzyme. Finally, no significant differences were detected concerning body weight, fresh organ weights and no sexual differences were detected.
The acreage for genetically modified crops (GMOs)—particularly soybean—has steadily increased since 1996, when the first crop of Roundup Ready soybean (intended for food production) was grown. The Roundup Ready soybean varieties derive from a soybean line into which a glyphosate-resistant enolpyruvylshikimate-3-phosphate-synthase (EPSPS) gene was introduced. The inserted and the flanking regions in Roundup Ready soybean have recently been characterized. It was shown that a further 250-bp fragment of the epsps gene is localized downstream of the introduced nos terminator of transcription, derived from the nopaline synthase gene from Agrobacterium tumefaciens. We examined whether this 250-bp fragment could be of functional importance. Our data demonstrate that at least 150bp of this DNA region are transcribed in Roundup Ready soybean. Transcription of the fragment depends on whether read-through events ignore the nos terminator signal located upstream. Our data also indicate that the read-through product is further processed, resulting in four different RNA variants from which the transcribed region of the nos terminator is completely deleted. Deletion results in the generation of open reading frames which might code for (as yet unknown) EPSPS fusion proteins. The nos terminator is used as a regulatory element in several other GMOs used for food production. This implies that read through products and transcription of RNA variants might be a common feature in these GMOs.
Pancreatitis-associated protein (PAP) is a secretory protein not normally expressed in healthy pancreas but highly induced during acute pancreatitis. While PAP has been shown to be anti-bacterial and anti-apoptotic in vitro, its definitive biological function in vivo is not clear.
To elucidate the function of PAP, antisense oligodeoxyribonucleotides (AS-PAP) targeting all three isoforms of PAP were administered via intrapancreatic injections (5 mg kg day, 2 days) to rats prior to induction of pancreatitis.
Severity of pancreatitis and cytokine gene expression in peripheral blood mononuclear cells (PBMC) were evaluated. Administration of AS-PAP, but not the scrambled oligodeoxyribonucleotide (SC-PAP) control, reduced pancreatitis-induced PAP expression by 55.2 +/- 6.4%, 44.0 +/- 8.9%, and 38.9 +/- 10.7% for PAP isoforms I, II, and III, respectively, compared to saline-treated controls (P < 0.05 for all). Inhibition of PAP expression significantly worsened pancreatitis: serum amylase activity, pancreas wet weight (reflecting edema), and serum C-reactive protein levels all increased in AS-PAP-treated animals compared to SC-PAP-treated controls (by 3.5-, 1.7-, and 1.7-fold, respectively; P < 0.05 for all). Histopathologic evaluation of pancreas revealed worsened edema, elevated leukocyte infiltration, and fat necrosis after AS-PAP treatment. Gene expressions of IL-1 microm and IL-4 were significantly higher in PBMC isolated from AS-PAP-treated rats compared to SC-PAP controls.
This is the first in vivo evidence indicating that PAP mediates significant protection against pancreatic injury. Our data suggest that PAP may exert its protective function by suppressing local pancreatic as well as systemic inflammation during acute pancreatitis.
Localized acute necrohemorrhagic pancreatitis was induced in rats by multiple trypsin injections. Morphological alterations were monitored by light and electron microscopy until complete recovery. In the acute phase, typical pictures of focal acute necrohemorrhagic pancreatitis were observed. In the postacute phase, fibrosis and tubular complexes are characteristic of damaged areas. Tubular complexes appear from the dedifferentiation of acinar cells. They are characterized by duct-like cells bordering wide, empty luminae. In the recovery phase, cellular proliferation was accompanied by differentiation, with progressive acquisition of the morphological characteristics of acinar cells at the periphery of the tubular complexes. In that instance, cellular proliferation was concomitant with the development of collagen septa in tubular complexes. In these structures both duct-like and acinar-like cells presented mitoses. Cell division persisted in the dedifferentiated cells until tubular complexes disappeared. A very similar process was observed in the embryonic pancreas, where organized parenchyma originated from proliferation and differentiation of protodifferentiated cells. We concluded that pancreatic repair following necrohemorrhagic pancreatitis involves proliferation of cells from intact acini and from tubular complexes, at variance with edematous pancreatitis, where regeneration is exclusively due to acinar cell proliferation.
Expression of the pancreatitis-associated protein I (PAP I), an exocrine pancreatic protein, increases rapidly and strongly in acinar cells during the acute phase of pancreatitis. This is reminiscent of the response to stress of acute phase proteins. We have previously demonstrated that serum factors from rats with acute pancreatitis, but not from healthy rats, could induce endogenous PAP I gene expression in the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C., Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204, 238-243). In the present work, we have evaluated the influence of several mediators of inflammation on rat PAP I gene transcription in these cells. Tumor necrosis factor alpha induced an increase in PAP I mRNA expression, and interferon gamma caused an even greater increase in PAP I mRNA level. These stimulations were antagonized by dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also ineffective. IL-6 and dexamethasone together induced a marked stimulation of PAP I gene transcription, and this effect was slightly attenuated by IL-1. To analyze the cis-regulatory elements responsible for the induction of transcription, we fused a 1.2-kilobase segment of the rat PAP I promoter to the chloramphenicol acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA was transfected into AR-42J cells. Addition of IL-6 or dexamethasone was ineffective, whereas their mixture increased the CAT activity 12 times. Progressive deletions of the PAP I promoter were then fused to the CAT gene, and the constructs were transfected to AR-42J cells. A 12-fold increase in CAT activity was seen upon IL-6/dexamethasone treatment with constructs containing more than 274 base pairs upstream from the cap site. In that region, two sequences are similar to the canonical IL-6 response element. Site-directed mutagenesis of these regions strongly decreased induction, showing that they were functional. PAP I should therefore be classified among acute phase proteins of class 2, whose expression is increased by IL-6 acting in combination with glucocorticoids.
The safety of 5-enolpyruvylshikimate-3-phosphate synthase enzyme derived from Agrobacterium sp. strain CP4 (CP4 EPSPS) was assessed. CP4 EPSPS is the only protein introduced by genetic manipulation that is expressed in glyphosate-tolerant soybeans, which are being developed to provide new weed-control options for farmers. Expression of this protein in plants imparts high levels of glyphosate tolerance. The safety of CP4 EPSPS was ascertained by evaluating both physical and functional characteristics. CP4 EPSPS degrades readily in simulated gastric and intestinal fluids, suggesting that this protein will be degraded in the mammalian digestive tract upon ingestion as a component of food or feed, There were no deleterious effects due to the acute administration of CP4 EPSPS to mice by gavage at a high dosage of 572 mg/kg body wt, which exceeds 1000-fold tha anticipated consumption level of food products potentially containing CP4 EPSPS protein. CP4 EPSPS does not pose any important allergen concerns because this protein does not possess characteristics typical of allergenic proteins. These data, in combination with seed compositional analysis and animal feeding studies, support the conclusion that glyphosate-tolerant soybean are as safe and nutritious as traditional soybeans currently being marketed.
Animal feeding studies were conducted with rats, broiler chickens, catfish and dairy cows as part of a safety assessment program for a soybean variety genetically modified to tolerate in-season application of glyphosate. These studies were designed to compare the feeding value (wholesomeness) of two lines of glyphosate-tolerant soybeans (GTS) to the feeding value of the parental cultivar from which they were derived. Processed GTS meal was incorporated into the diets at the same concentrations as used commercially; diary cows were fed 10 g/100 g cracked soybeans in the diet, a level that is on the high end of what is normally fed commercially. In a separate study, laboratory rats were fed 5 and 10 g unprocessed soybean meal 100 g diet. The study durations were 4 wk (rats and dairy cows), 6 wk (broilers) and 10 wk (catfish). Growth, feed conversion (rats, catfish, broilers), fillet composition (catfish), and breast muscle and fat pad weights (broilers) were compared for animals fed the parental and GTS lines. Milk production, milk composition, rumen fermentation and nitrogen digestibility were also compared for dairy cows. In all studies, measured variables were similar for animals fed both GTS lines and the parental line, indicating that the feeding value of the two GTS lines is comparable to that of the parental line. These studies support detailed compositional analysis of the GTS seeds, which showed no meaningful differences between the parental and GTS lines in the concentrations of important nutrients and antinutrients. They also confirmed the results of other studies that demonstrated the safety of the introduced protein, a bacterial 5-enolpyruvyl-shikimate-3-phosphate synthase from Agrobacterium sp. strain CP4.
A 38-d feeding study evaluated whether standard broiler diets prepared with transgenic Event 176-derived "Bt" corn (maize) grain had any adverse effects on male or female broiler chickens as compared to diets prepared with nontransgenic (isogenic) control corn grain. No statistically significant differences in survival or BW were observed between birds reared on mash or pelleted diets prepared with transgenic corn and similar diets prepared using control corn. Broilers raised on diets prepared from the transgenic corn exhibited significantly better feed conversion ratios and improved yield of the Pectoralis minor breast muscle. Although it is not clear whether this enhanced performance was attributable to the transgenic corn per se, or due to possible slight differences in overall composition of the formulated diets, it was clear that the transgenic corn had no deleterious effects in this study.
A genetically modified Bt176 corn hybrid (Rh208Bt)--providing control of European corn borer damage--and the conventional isogenic hybrid (Rh208)--harvested as whole plant silage--were evaluated in three separate feeding trials to verify that the in vivo feeding value was substantially equivalent among modified and conventional hybrids. In the first trial, after a week of preexperiment, two sets of six Texel sheep, housed in digestibility crates, were fed silage sources of Rh208 and Rh208Bt hybrids, and silage of three additional control varieties of low, intermediate, and high feeding value (Rh289, Adonis, and Adonis bm3) for 1 wk. Feed offered to sheep was adjusted to maintenance requirements based on metabolic body weight. Agronomic and biochemical traits were similar among the Rh208 and Rh208Bt hybrids. Organic matter digestibility (67.1 and 67.6%), crude fiber digestibility (52.9 and 54.2%), and neutral detergent fiber digestibility (50.2 and 49.0%) were not significantly different among Rh208 and Rh208Bt hybrids. In the second trial, two sets of 24 Holstein cows were fed silage from Rh208 and Rh208Bt corn hybrids for 13 wk, 9 wk after calving, and including 2 wk of preexperiment. Fat-corrected milk yield (31.3 and 31.4 kg/d), protein content (31.7 and 31.6 g/kg) and fat content (36.7 and 37.0 g/kg) in milk of dairy cows were unaffected by hybrid source. Body weight gains of cattle were not different. However, intake was significantly higher in cows fed Rh208Bt silage. In the third trial, five midlactation multiparous Holstein cows were successively fed the silage from Rh208 and Rh208Bt corn hybrids 2 or 3 wk. Data were considered only for the last week of each period. There were no significant effects on protein fractions, fatty acid composition, or coagulation properties of milk between Rh208 and Rh208Bt fed cattle. Cattle and sheep can perform equally well with a conventional or a genetically modified Bt176 corn silage.
A feeding study evaluated whether standard broiler diets prepared with grain derived from Syngenta Seeds NK Brand Bacillus thuringiensis (Bt) Corn hybrids had any adverse effects on male or female broiler chickens. Four kinds of corn grain were used in this study: (1) grain from the Bt-expressing field corn hybrid N7070Bt, (2) grain from the N7070Bt hybrid that had been sprayed with Liberty brand herbicide (glufosinate) according to manufacturer's instructions (N7070Bt + Liberty), (3) grain from standard N7070 (non-Bt isoline of N707OBt) grain, and (4) a lot of North Carolina grown grain from the 2000 growing season (NC2000). The amino acid balance for the four lots of corn was similar relative to their crude protein content; however, the NC2000 corn had higher protein content. Diets with the higher protein NC2000 season corn were amended with a combination of sand, ground cardboard (Solka Floc), and poultry fat so that the metabolizable energy and crude protein content of the diluted diets would be similar to that of the isoline and transgenic diets. Growth of broilers was excellent with males being significantly heavier than females (2,497 g vs. 2,103 g) at 42 d of age. BW of live birds at 42 d was within 26 g for the three treatment groups fed corn that was from the same genetic background, i.e., the two Bt transgenic groups (N7070Bt, N7070Bt + Liberty), and the non-Bt N7070 isoline corn group, while BW for the NC2000 group was significantly lower by 93 g. There was no overall corn source effect on feed conversion ratio (FCR) among the isoline and transgenic corn sources to 42 d of age, but FCR was poorer for broilers consuming the commercial NC2000 corn. There was no overall effect of corn source on survivability to 42 d. Carcass analysis at 48 d demonstrated no differences in percentage carcass yield due to corn source among males and females. The transgenic N7070Bt and N7070Bt + Liberty hybrid diets supported excellent broiler chicken growth with mortality and FCR that were similar to that supported by the N7070 isoline control and better than rates from the commercial NC2000 corn without significant differences among treatment groups in carcass yield. It was clear that the transgenic corn had no deleterious or unintended effects on production traits of broiler chickens in this study.
Lactating dairy cows were used to determine effects of feeding glyphosate-tolerant or insect-protected corn hybrids on feed intake, milk production, milk composition, and ruminal digestibility. Corn resistant to European corn borer (Ostrinia nubilalis) infestation (Bt-MON810), or its nontransgenic control (Bt-CON), were planted in alternating fields during two successive years. One-half of each strip was harvested for whole plant corn silage and the remainder was allowed to mature and harvested as grain. Effects of feeding diets containing either Bt-MON810 or Bt-CON grain and silage were determined in two experiments (1 and 2) conducted during successive years. In experiment 3, glyphosate-tolerant Roundup Ready corn (RR-GA21) or its nontransgenic control (RR-CON) corn were grown in alternating fields during one cropping season. Diets contained 42 to 60% corn silage and 20 to 34% corn grain from Bt-MON810, RR-GA21, or the appropriate nontransgenic counterpart; treatments were applied using a switchback design. Cows were fed ad libitum and milked twice daily. There were no differences for nutrient composition between silage sources or between grain sources within an experiment. Data for experiments 1 and 2 indicated similar dry matter intake (DMI), 4% fat-corrected milk (FCM) production, and milk composition between Bt-MON810 and Bt-CON diets. There were no differences for DMI, 4% FCM production, and milk composition between RR-GA21 and RR-CON diets. There was no difference in ruminal degradability, determined separately for corn silage and corn grain, for RR-GA21 or Bt-MON810-hybrids compared with their respective controls. These data demonstrate equivalence of nutritional value and production efficiency for corn containing Bt-MON810 compared with its control and for RR-GA21 corn compared with its control.
Sixteen multiparous Holstein cows averaging 74 d in milk were used in a replicated 4 x 4 Latin square to compare the effects on animal performance of feeding whole plant silage and grain from a glyphosate-tolerant corn hybrid (event NK603), a nontransgenic control hybrid, and two commercial nontransgenic hybrids (DK647 and RX740). The grain and silage from the four corn hybrids were produced using the same procedures and under similar agronomic conditions at the University of Illinois. On a dry matter (DM) basis, diets contained 30% corn silage and 27.34% corn grain produced either from event NK603, a nontransgenic control, or commercial hybrids. Apart from the DM content of silages, the chemical composition of both grain and silage produced from the four corn hybrids were substantially equivalent. Feeding diets that contained event NK603 and DK647 hybrids tended to decrease DM intake (DMI) compared with the control nontransgenic and RX740. The intakes of crude protein (CP), acid and neutral detergent fiber, and nonfiber carbohydrates were not different for cows fed event NK603 and control diets. The RX740 diet resulted in the highest intakes of fiber and CP, whereas the DK647 diet resulted in the lowest intake of CP. These differences in nutrient intake arose from small variations in both the DMI and the chemical composition of feed ingredients and experimental diets. Production of milk and 3.5% fat-corrected milk; milk fat, CP, and true protein percentage and yield; milk urea N; milk total solids percentage and yield; and somatic cell count were not affected by treatments. These data indicate that the stable insertion of the gene that confers tolerance to glyphosate (event NK603) in the corn line used in this experiment does not affect its chemical composition and nutritional value for lactating dairy cows when compared with conventional corn.
We carried out ultrastructural morphometrical and immunocytochemical analyses on pancreatic acinar cell nuclei from mice fed on genetically modified (GM) soybean, in order to investigate possible structural and molecular modifications of nucleoplasmic and nucleolar constituents. We found a significant lowering of nucleoplasmic and nucleolar splicing factors as well as a perichromatin granule accumulation in GM-fed mice, suggestive of reduced post-transcriptional hnRNA processing and/or nuclear export. This is in accordance to already described zymogen synthesis and processing modifications in the same animals.
Isoflavones are biologically active compounds occurring naturally in a variety of plants, with relatively high levels found in soybeans. Twelve laboratories participated in a collaborative study to determine the aglycon isoflavone content of 8 test samples of soy and foods containing soy. The analytical method for the determination of isoflavones incorporates a mild saponification step that reduces the number of analytes measured and permits quantitation versus commercially available, stable reference standards. Test samples were extracted at 65°C with methanol–water (80 + 20), saponified with dilute sodium hydroxide solution, and analyzed by reversed-phase liquid chromatography with UV detection at 260 nm. Isoflavone results were reported as μg/aglycon/g or μg aglycon equivalents/g. The 8 test samples included 2 blind duplicates and 4 single test samples with total isoflavone concentrations ranging from approximately 50 to 3000 μg/g. Test samples of soy ingredients and products made with soy were distributed to collaborators with appropriate reference standards. Collaborators were asked to analyze test samples in duplicate on 2 separate days. The data were analyzed for individual isoflavone components, subtotals of daidzin–daidzein, glycitin–glycitein, and genistin–genistein, and total isoflavones. The relative standard deviation (RSD) for repeatability was 1.8–7.1%, and the RSD for reproducibility was 3.2–16.1% for total isoflavone values of 47–3099 μg/g.
AIM: To establish a non-traumatic, easy to induce and reproducible mouse model of severe acute pancreatitis (SAP) induced with caerulein and lipopolyasccharide (LPS). METHODS: Thirty-two healthy mature NIH female mice were selected and divided at random into four groups (each of 8 mice), i.e., the control group (NS group), the caerulein group (Cn group), the lipopolysaccharide group (LPS group), and the caerulein+LPS group (Cn+LPS group). Mice were injected intraperitoneally with caerulein only, or LPS only, and caerulein and LPS in combination. All the animals were then killed by neck dislocation three hours after the last intraperitoneal injection. The pancreas and exo-pancreatic organs were then carefully removed for microscopic examination. And the pancreatic acinus was further observed under transmission electron microscope (TEM). Pancreatic weight, serum amylase, serum nitric oxide (NO) concentration, superoxide dismutase (SOD) and malondialdehyde (MDA) concentration of the pancreas were assayed respectively. RESULTS: (1) NS animals displayed normal pancreatic structure both in the exocrine and endocrine. In the LPS group, the pancreas was slightly edematous, with the infiltration of a few inflammatory cells and the necrosis of the adjacent fat tissues. All the animals of the Cn group showed distinct signs of a mild edematous pancreatitis characterized by interstitial edema, infiltration of neutrophil and mononuclear cells, but without obvious parenchyma necrosis and hemorrhage. In contrast, the Cn+LPS group showed more diffuse focal areas of nonviable pancreatic and hemorrhage as well as systemic organ dysfunction. According to Schmidt's criteria, the pancreatic histologic score showed that there existed significant difference in the Cn+LPS group in the interstitial edema, inflammatory infiltration, parenchyma necrosis and parenchyma homorrhage in comparison with those of the Cn group, LPS group and NS group (P<0.01 or P<0.05). (2) The ultrasturcture of acinar cells was seriously damaged in the Cn+LPS group. Chromatin margination of nuclei was present, the number and volume of vacuoles greatly increased. Zymogen granules (ZGs) were greatly decreased in number and endoplasmic reticulum exhibited whorls. The swollen mitochondria appeared, the crista of which was decreased in number or disappeared. (3) Pancreatic weight and serum amylase levels in the Cn+LPS was significantly higher than those of the NS group and the LPS group respectively (P<0.01 or P<0.05). However, the pancreatic wet weight and serum amylase concentration showed no significant difference between the Cn+LPS group and the Cn group. (4) NO concentration in the Cn+LPS group was significantly higher than that of NS group, LPS group and Cn group(P<0.05 or P<0.01). 5) The SOD and MDA concentration of the pancreas in the Cn+LPS group were significantly higher than those of NS, LPS and Cn groups (P<0.05 or P<0.01). CONCLUSION: The mouse model of severe acute pancreatitis could be induced with caerulein and LPS, which could be non-traumatic and easy to induce, reproducible with the same pathological characteristics as those of SAP in human, and could be used in the research on the mechanism of human SAP.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
An improved method was developed to determine active soybean lectin in processed foods containing soybean. Freeze drying of crude extracts followed by affinity chromatography allowed the authors to purify and quantitate active lectin. After dialysis, concentrated extracts from products were loaded on to a chromatography column of immobilized N-acetyl-galactosamine. Active lectin content of soybean flours depended on the processing method used. The highest level found was for raw seeds (3600 μg/g), and the lowest for texturized flour (12·9 μg/g). Direct human consumption foods contained various lectin concentrations. While meat substitutes were free of active lectin, milk substitutes, and bakery products had low levels. A cereal type food and a cookie showed the highest lectin concentrations (187·2 and 24·2 μg/g, respectively).
We compared the effect of high protein diets enriched with casein, fish, or soybean on enzyme content and mRNA levels in pancreata of postweaning and adult rats of two different strains. In the first experiment, 72 male Fischer rats (age 5 weeks) were divided into six groups and fed with one of six diets containing 20% or 50% protein as fish meal, casein, or soybean for 1 or 3 weeks. In a second experiment, 36 Fischer and 36 Wistar rats were divided into six groups and fed with the same diets for 1 week. In both experiments, rats were sacrificed at the end of the experimental feeding period and pancreata were excised and prepared for biochemical assay and mRNA extraction. Activities and mRNA levels were determined for each enzyme (amylase, lipase, chymotrypsin, trypsin, and elastases). Pancreas weight and its total protein content were modulated by the amount of dietary protein and duration of diet. These parameters were significantly different in Fischer and Wistar rats. In the latter strain, the nature of dietary protein also influenced pancreas weight. In Fischer rats, amylase specific activity was decreased after feeding 50% casein diet for 1 or 3 weeks and 50% fish or soybean diets for 3 weeks. The decrease of specific mRNA was more pronounced after a 3-week than after a 1-week feeding, suggesting that transcriptional regulation replaced progressively a translational one. In Wistar rats, amylase specific activity was not modified, but mRNAs were decreased after feeding high-protein diets. Lipase specific activity and mRNAs were not modified by any diet in any group. Chymotrypsin specific activity was increased after feeding 50% casein and soybean diets for 1 week and any 50% protein diet for 3 weeks. This effect was more pronounced in adult rats fed high protein diets for 1 week. In young Fischer rats, chymotrypsinogen mRNAs were increased after feeding 50% casein diets: in adult rats of both strains this parameter was increased by all diets except when the 50% soybean diet was provided to Wistar. Trypsin specific activity was increased after feeding 50% casein diet for 1 week and 50% fish or soybean diet for 3 weeks in Fischer rats but not altered in Wistar. The expression of trypsinogen mRNA was only increased after feeding 50% casein diet to both strains and 50% soybean diet to Wistar rats, suggesting that the regulation of its expression is different with the nature of protein. Elastase specific activities were increased by high-protein diets in both strains, but this effect was more pronounced in Wistar rats: these enzyme mRNAs were not altered, suggesting that the regulation was translational. In conclusion, it appears that the kinetics of adaptation of enzymes is different depending on the nature of dietary protein and the strain of rats used in the experiment. Amylase biosynthesis is regulated at the transcriptional level. Chymotrypsinogen and trypsinogen mRNA level showed that casein-induced adaptation was modulated via transcription, while other diets induced adaptation via postranscriptional events. Elastases adapt differently depending on the nature of the protein and the regulation of their expression is mostly posttranscriptional.
The amounts of 12 isoflavones were measured in 8 American and 3 Japanese soybean varieties by using C-18 reversed-phase high-performance liquid chromatography. In Vinton 81 soybeans of 1989-1991, variation in total isoflavone ranged from 1176 to 3309 mu g/g. Isoflavone amounts of soybeans grown in different locations in 1991 ranged from 1176 to 1749 mu g/g. Crop year seemed to have a much greater influence on isoflavone content in Vinton 81 than did location. Isoflavones in the other seven American varieties (Pioneer 9111, Pioneer 9202, Prize, HP204, LS301, XL72, Strayer 2233) ranged from 2053 to 4216 mu g/g. The major isoflavone constituents were 6''-O-malonylgenistin, genistin, 6''-O-malonyldaidzin, and daidzin. Isoflavone contents in three Japanese varieties (Keburi, Kuro diazu, Raiden) ranged from 2041 to 2343 mu g/g and from 1261 to 1417 mu g/g for soybeans grown in 1991 and 1992, respectively. Compared with American varieties, Japanese varieties had higher 6''-O-malonylglycitin contents and higher ratios of 6''-O-malonyldaidzin to daidzin and 6''-O-malonylgenistin and genistin.
The influence of transgenic event CBH 351 (StarLink; SL)-derived hybrid corn on the growth performance, health condition and physiological function in broiler chicks, as well as the possible transfer of the cry9C gene and Cry9C protein to blood, liver and muscle were examined in comparison with chicks fed on a diet with non-transgenic corn (SL-F). Bodyweight gain and feed conversion ratio in the chicks fed on a diet with SL were significantly greater than in chicks fed on a diet with SL-F during the starter phase (0–3 weeks of age), but this significant difference disappeared during the finisher phase (4–7 weeks of age). No abnormalities in health condition in either SL or SL-F groups were observed, and livability did not differ significantly between SL and SL-F groups. Moreover, no significant differences in serum biochemical and hematological values, histopathological observation and necropsy findings were observed between SL and SL-F groups at the end of the experiment. The cry9C gene and Cry9C protein were not detected in blood, liver and muscle of chicks at 3, 5 or 7 weeks of age. The results indicate that feeding SL does not influence growth performance, health condition or physiological function in broiler chicks, and the cry9C gene and Cry9C protein are not transferred to the blood, liver and muscles of broilers.
Ischemia has been considered to play a role in the development of acute pancreatitis. The aim of this study was to investigate the effect of ischemia, caused by hemorrhagic shock, on cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by the intravenous infusion of a supramaximally stimulating dose of cerulein (10 g/kg/hr) for 6 hr. Hemorrhagic shock was induced by the removal of blood until the mean arterial blood pressure reached 35 mm Hg. This level was maintained for 30 min, after which time all the blood was reinfused. Hemorrhagic shock alone induced no morphological change in the pancreas. However, after the induction of hemorrhagic shock in animals treated with cerulein, hemorrhage and parenchymal necrosis were frequently observed in the pancreas. Seven of 20 rats (35%) receiving cerulein plus hemorrhagic shock had died by 48 hr after the start of cerulein infusion, whereas none of the rats in the cerulein or shock group died during this experiment. Cathepsin B activity in the pancreas of the cerulein plus shock group was significantly higher than in the other groups at 48 hr. These results suggest that ischemia may be a contributing factor in the pathogenesis of acute pancreatitis.
The surveillance of food labelling concerning genetically modified organisms (GMOs) requires DNA-based analytical techniques.
Present assay systems allow the detection of GMO in food; however, they do not permit their quantitation. In this study, we
report the development of quantitative competitive polymerase chain reaction (QC-PCR) systems for the detection and quantitation
of the Roundup Ready soybean (RRS) and the Maximizer maize (MM) in food samples. Three DNA fragments that differ from the
GMO-specific sequences by an insertion were constructed and used as internal standards in the PCR. These standards were calibrated
by co-amplifying with mixtures containing RRS DNA and MM DNA, respectively. The calibrated QC-PCR systems were applied to
nine commercial food samples containing RRS DNA and to three certified RRS flour mixtures in order to elucidate whether these
food samples contain more or less than 1% RRS DNA. Finally, the GMO contents of four samples that were found to contain more
than 1% RRS were determined by QC-PCR using various amounts of standard DNA.
Various novel biochemical markers indicate pancreatic cellular injury more accurately than serum amylase or lipase. One of these is a non-enzymatic secretory protein called pancreatitis-associated protein (PAP).The main function of PAP is unclear at present but it may be an acute phase protein in the defence reactions of pancreatic cells. The protein was characterized in 1984 as a serum marker of pancreatitis. The serum PAP is expressed 6 hours after the induction of pancreatitis, and it increases to maximal levels within 2-4 days: PAP is not sufficiently sensitive for the diagnosis of acute pancreatitis in the emergency room. The sensitivity and specificity of PAP in the differentiation of severe from mild pancreatitis is between 60-70%. This is not superior to serum CRP assays or CT scans. PAP increases in pancreatic cellular injury without pancreatitis (subclinical cell damage, graft rejection) where PAP may have a diagnostic role.
Ischemia has been considered to play a role in the development of acute pancreatitis. The aim of this study was to investigate the effect of ischemia, caused by hemorrhagic shock, on cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by the intravenous infusion of a supramaximally stimulating dose of cerulein (10 micrograms/kg/hr) for 6 hr. Hemorrhagic shock was induced by the removal of blood until the mean arterial blood pressure reached 35 mm Hg. This level was maintained for 30 min, after which time all the blood was reinfused. Hemorrhagic shock alone induced no morphological change in the pancreas. However, after the induction of hemorrhagic shock in animals treated with cerulein, hemorrhage and parenchymal necrosis were frequently observed in the pancreas. Seven of 20 rats (35%) receiving cerulein plus hemorrhagic shock had died by 48 hr after the start of cerulein infusion, whereas none of the rats in the cerulein or shock group died during this experiment. Cathepsin B activity in the pancreas of the cerulein plus shock group was significantly higher than in the other groups at 48 hr. These results suggest that ischemia may be a contributing factor in the pathogenesis of acute pancreatitis.
We investigated pancreatic gene expression in the rat in response to taurocholate-induced acute pancreatitis. Concentrations of transcripts encoding pancreatic protein showed noncoordinated alterations. Contents in amylase, trypsinogen I, chymotrypsinogen B, elastase 1, and procarboxypeptidase A mRNAs decreased by greater than 50% during the acute phase (days 0-2), whereas actin and lithostathine mRNAs increased 5 and 0.6 times, respectively, and pancreatitis-associated protein (PAP) mRNA increased greater than 200 times, indicating redirection of the pattern of gene expression. Synthesis of pancreatic proteins was also altered in a noncoordinated manner. During the acute phase, it decreased more for trypsinogen I and chymotrypsinogen B than for amylase and lipase, whereas synthesis of the PAP increased dramatically. For amylase and chymotrypsinogen B, we compared the patterns of changes in mRNA concentrations, rates of synthesis, and pancreatic contents. Changes in enzyme contents and synthetic rates were temporally correlated during the acute phase. On the contrary, changes in mRNA concentrations and enzyme synthesis were not coordinated, suggesting that control of synthesis partly occurred at the posttranscriptional level. It was concluded that induction of pancreatitis is accompanied by transcriptional and posttranscriptional modifications resulting in rapid and massive rearrangement of the pattern of pancreatic protein gene expression.
Cholecystokinin-pancreozymin (CCK-PZ) is involved in the regulation of pancreatic protein synthesis and secretion. We demonstrate here that CCK-PZ also stimulates RNA synthesis. Rats were killed 0, 30, 60, 120, 240 or 480 min after intraperitoneal injection of CCK-PZ (8 U/kg). Nuclei were prepared from pancreata and used for in vitro RNA synthesis ('run-on' experiments) in the presence of [alpha-32P]UTP. Total RNA synthesis increased after CCK-PZ with maximum UTP incorporation at 60 min. Contributions of RNA polymerase II, responsible for mRNA synthesis, and RNA polymerases I and III could be separately estimated by using alpha-amanitin. RNA polymerase I and III activities increased by 68% after CCK-PZ, whereas RNA polymerase II activity increased by 113%. Rates of synthesis of amylase, chymotrypsinogen B, trypsinogen I and actin mRNAs were estimated by quantitative hybridization of newly synthesized transcripts to specific cDNA clones. Synthesis of the four RNAs increased with a maximum between 60 and 120 min after CCK-PZ, stimulation being more important for the serine proteinases than for amylase. It was concluded that, in rat pancreas, CCK-PZ controls gene expression at the transcriptional level.
The changes in plasma and duodenal cholecystokinin (CCK) concentrations after pancreatic duct occlusion were examined in rats. The rats were sacrificed 1, 3, 7, 10, 14, and 30 days after occlusion of the duct. Histological examination showed acute inflammation on days 1 and 3 after duct occlusion, interstitial fibrosis and regenerative changes on days 7, 10, and 14, and pancreatic atrophy on day 30. The plasma CCK concentration increased from 0.45 pM to 2.0 pM after the occlusion and then remained high throughout the observation period. In contrast to the stable increase in plasma CCK concentration, the CCK content in the duodenum increased on days 1 and 3, decreased on day 7, increased on day 10, reaching over the control level on day 14, and then returned to the control level on day 30. Administration of boiled and 10-fold concentrated rat pancreatic juice or human pancreatic secretory trypsin inhibitor for seven days after pancreatic duct occlusion reversed the decrease in duodenal CCK content. The major molecular forms of duodenal CCK were CCK-8, -33, and -58. These results indicate that (1) basal plasma CCK concentration did not reflect the duodenal CCK content, (2) duodenal CCK content was well correlated with a decrease in inflammation in the pancreas, and (3) a nonenzymatic component in the pancreatic juice reversed the decrease in duodenal CCK content and body weight caused by pancreatic duct occlusion.
In the current study two monoclonal antibodies (mAb) were used to investigate the expression of adult acinar and duct cell-specific antigens and their relationship with cell growth in primary acinar cell cultures. We have previously found that adult mouse pancreatic acinar cells divide in primary culture. Furthermore, during growth the cells lose their differentiated morphology and exhibit decreased expression of secretory proteins, followed by some degree of morphological redifferentiation after reaching confluency. A mAb specific in the adult pancreas for acinar cells (mAb Acinar-1) and another specific in the adult pancreas for duct cells (mAb Duct-1) were generated using such cultures as the immunogen. The starting material for the cultures consisted of predominantly Acinar-1 positive cells which incorporated [3H]thymidine, as determined by autoradiography and immunofluorescence labeling. However, expression of the acinar antigen persisted for only the first 3 to 7 days in culture. By contrast, expression of the duct antigen was rare until after 5 days in culture and was highest at day 9, the peak of cell growth. Dual label immunofluorescence showed that during the growth phase fewer cells expressed the acinar antigen, most expressed the duct antigen, and occasional cells expressed both antigens. After reaching confluency, the growth rate declined from days 15 to 21, and the cells progressively regained the acinar antigen with a concomitant loss of the duct antigen. mAb labeling was morphometrically quantitated and showed that more than 97% of the labeled area was Acinar-1 positive at 3 days, which decreased to approximately 16% at day 9, and then returned to over 97% by day 21 of culture. Ultrastructural immunolabeling showed that Acinar-1 positive cells at 21 days had well organized rough endoplasmic reticulum and small apical vesicles, while Duct-1 positive cells were undifferentiated in appearance (day 9) or had numerous mitochondria (day 21). Thus, changes in cell-specific antigens were paralleled by cell type associated morphological characteristics and indicate that adult acinar cells can retrodifferentiate to a more duct-like cell while retaining the potential to express an acinar-specific antigen.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Soybeans are high in protein but also contain a number of minor constituents traditionally considered to be antinutritional factors. These include trypsin inhibitors, phytic acid, saponins and isoflavones. These compounds are now thought to have beneficial biological effects in the diet, such as lowering blood cholesterol or preventing cancer. Soybean processing changes the content of these minor constituents in various ways. This review discusses the changes in content of trypsin inhibitors, phytic acid, saponins and isoflavones as soybeans are processed into the conventional protein ingredients, flours, concentrates and isolates, as well as some of the traditional Oriental soybean foods.
It has been assumed in the past that pancreatic acinar cells represent an irreversible end stage in development. Consequently, when there was an increase in structures that had the morphology of ductules, the interpretation was that they were derived from the proliferation of stem cells and/or pre-existing ductular cells. Pancreatitis, however, is regressive in nature [Bockman (1984) In: Pancreatitis: Concepts and Classification. Gyr, K.E., Singer, M.V., Sarles, H., eds. Elsevier, Amsterdam, pp. 11-15]. That is, it is characterized by parenchymal destruction and loss, rather than by expansion of parenchyma. Furthermore, it was assumed that the organization of the pancreatic parenchyma is like bunches of grapes, with spheroidal acini representing the grapes, and the ductules representing the stems. Given this organization, it would be difficult to understand how regressive changes could lead to clusters of ductular structures. Investigations using three-dimensional reconstruction and retrograde injections have altered our idea of pancreatic organization. In addition to spheroidal acini, there also are other shapes, including tubular acini. Moreover, ductules do not necessarily stop when they encounter an acinus. They may emerge on the other side. Combined ductular and acinar lumina may anastomose with each other. It is now clear that pancreatic acini may undergo redifferentiation, taking on the morphology of ductules and forming tubular complexes during pancreatitis, as well as in response to pancreatic cancer, cystic fibrosis, or blockage of the ductal system. With this understanding of pancreatic architecture and morphological plasticity, it is easier to understand the changes one sees with pancreatic diseases.
The present work has been designed to study the effect of feeding on transgenic potatoes, which carry the CryI gene of Bacillus thuringiensis var. kurstaki strain HD1, on the light and electron microscopic structure of the mice ileum, in comparison with feeding on potatoes treated with the 'delta-endotoxin' isolated from the same bacterial strain. The microscopic architecture of the enterocytes of the ileum of both groups of mice revealed certain common features such as the appearance of mitochondria with signs of degeneration and disrupted short microvilli at the luminal surface. However, in the group of mice fed on the 'delta-endotoxin', several villi appeared with an abnormally large number of enterocytes (151.8 in control group versus 197 and 155.8 in endotoxin and transgenic-treated groups, respectively). Fifty percent of these cells were hypertrophied and multinucleated. The mean area of enterocyte was significantly increased (105.3 microm(2) in control group versus 165.4 microm(2) and 116.5 microm(2) in endotoxin and transgenic-treated groups, respectively). Several forms of secondary lysosomes or auotophagic vacuoles were recognized in these cells. These changes were confirmed with the scanning electron microscope which revealed a remarkable increase in the topographic contour of enterocytes (23 microm in control group versus 44 microm and 28 microm in endotoxin and transgenic-treated groups, respectively) at the divulged surface of the villi. The basal lamina along the base of the enterocytes was damaged at several foci. Several disrupted microvilli appeared in association with variable-shaped cytoplasmic fragments. Some of these fragments contained endoplasmic reticulum, as well as ring-shaped annulate lamellae. In addition, the Paneth cells were highly activated and contained a large number of secretory granules. These changes may suggest that delta-endotoxin-treated potatoes resulted in the development of hyperplastic cells in the mice ileum. Although mild changes are reported in the structural configuration of the ileum of mice fed on transgenic potatoes, nevertheless, thorough tests of these new types of genetically engineered crops must be made to avoid the risks before marketing.
Diets containing genetically modified (GM) potatoes expressing the lectin Galanthus nivalis agglutinin (GNA) had variable effects on different parts of the rat gastrointestinal tract. Some effects, such as the proliferation of the gastric mucosa, were mainly due to the expression of the GNA transgene. However, other parts of the construct or the genetic transformation (or both) could also have contributed to the overall biological effects of the GNA-GM potatoes, particularly on the small intestine and caecum.
Feeding studies of transgenic potatoes with native and designed soybean glycinins in rats were done for four weeks. The designed glycinin has four additional methioninyl residues in the middle of the glycinin molecule. Rats were divided into four groups fed (I) only a commercial diet, (II) the diet plus non-transgenic potatoes, (III) the diet plus transgenic potatoes with native glycinin, and (IV) the diet plus transgenic potatoes with designed glycinin. Rats were fed 2,000 mg/kg-weight potatoes every day by oral administration. During the period tested, rats in each group (groups II, III, and IV) grew well without marked differences in appearance, food intake, body weight, or in cumulative body weight gain. No significant differences were also found in blood count, blood composition, and in internal organ weights among the rats after feeding potatoes (groups II, III, and IV) for four weeks. Necropsy at the end of experiment indicated neither pathologic symptoms in all rats tested nor histopathological abnormalities in liver and kidney. Judging from these results, the transgenic potatoes with glycinins are confirmed to have nearly the same nutritional and biochemical characteristics as non-transgenic one.
Infusion of sulphated cholecystokinin-8 (CCK-8S) in rats transiently increased the proliferation of pancreatic acinar cells, whereas the CCK-A receptor antagonist devazepide decreased such proliferation. This effect ceased after 3 days. CCK-8S or devazepide injected twice daily induced a persistent effect on the cell proliferation involving the major cells of the exocrine pancreas. The aim of this study was to examine the effect of continuous infusion of CCK-8S and devazepide on CCK-A receptor gene expression.
Male Sprague-Dawley rats received subcutaneous continuous infusion of 5 microg/kg/h CCK-8S, 200 microg/kg/h devazepide, or 1% bovine serum albumin (BSA) by means of osmotic minipumps. The rats were killed after 4 days; I h before being killed they received 5-bromo-2-deoxyuridine (BrdU) intraperitoneally. Plasma was collected for analysis of CCK. The pancreas was dissected, and indirect immunofluorescence for BrdU and CCK-A receptor was performed. In situ hybridization to CCK-A receptor mRNA was performed for examination and semiquantification of receptor gene expression.
Continuous infusion of CCK-8S led to a sixfold increase in plasma CCK and a 40% increase in pancreatic weight. Devazepide did not affect the CCK level but decreased the pancreatic weight by 24% compared with BSA-infused rats. The BrdU labeling indicated that CCK-8S had no effect on cell proliferation. Immunofluorescence for the CCK-A receptor showed a decreased labeling intensity after CCK-8S infusion. The mean optical density of in situ hybridization labeling of the sections from CCK-8S-treated rats was decreased to 37% +/- 3% of that in controls. Devazepide did not affect the CCK-A receptor gene expression.
Continuous stimulation of the CCK-A receptor led to a downregulation of the receptor gene expression in pancreatic acinar cells and decreased labeling of the receptor at immunohistochemistry. The results suggest that down-regulation of the receptor is a protective mechanism against overstimulation.
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way
to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform,
provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification
of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters
into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to
a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated
only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model
needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA
integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler
PCR using the established mathematical model.
Concerns have been raised about the potential effects of transgenic introductions on the genetic diversity of crop landraces and wild relatives in areas of crop origin and diversification, as this diversity is considered essential for global food security. Direct effects on non-target species, and the possibility of unintentionally transferring traits of ecological relevance onto landraces and wild relatives have also been sources of concern. The degree of genetic connectivity between industrial crops and their progenitors in landraces and wild relatives is a principal determinant of the evolutionary history of crops and agroecosystems throughout the world. Recent introductions of transgenic DNA constructs into agricultural fields provide unique markers to measure such connectivity. For these reasons, the detection of transgenic DNA in crop landraces is of critical importance. Here we report the presence of introgressed transgenic DNA constructs in native maize landraces grown in remote mountains in Oaxaca, Mexico, part of the Mesoamerican centre of origin and diversification of this crop.
Isoflavones are biologically active compounds occurring naturally in a variety of plants, with relatively high levels found in soybeans. Twelve laboratories participated in a collaborative study to determine the aglycon isoflavone content of 8 test samples of soy and foods containing soy. The analytical method for the determination of isoflavones incorporates a mild saponification step that reduces the number of analytes measured and permits quantitation versus commercially available, stable reference standards. Test samples were extracted at 65 degrees C with methanol-water (80 + 20), saponified with dilute sodium hydroxide solution, and analyzed by reversed-phase liquid chromatography with UV detection at 260 nm. Isoflavone results were reported as microg/aglycon/g or microg aglycon equivalents/g. The 8 test samples included 2 blind duplicates and 4 single test samples with total isoflavone concentrations ranging from approximately 50 to 3000 microg/g. Test samples of soy ingredients and products made with soy were distributed to collaborators with appropriate reference standards. Collaborators were asked to analyze test samples in duplicate on 2 separate days. The data were analyzed for individual isoflavone components, subtotals of daidzin-daidzein, glycitin-glycitein, and genistin-genistein, and total isoflavones. The relative standard deviation (RSD) for repeatability was 1.8-7.1%, and the RSD for reproducibility was 3.2-16.1% for total isoflavone values of 47-3099 microg/g.
Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. In this article, protein- and DNA-based methods employing western blots, enzyme-linked immunosorbant assay, lateral flow strips, Southern blots, qualitative-, quantitative-, real-time- and limiting dilution-PCR methods, are discussed. Where information on modified gene sequences is not available, new approaches, such as near-infrared spectrometry, might tackle the problem of detection of non-approved genetically modified (GM) foods. The efficiency of screening, identification and confirmation strategies should be examined with respect to false-positive rates, disappearance of marker genes, increased use of specific regulator sequences and the increasing number of GM foods.
There are conflicting reports on the effect of soy and its components on mammary carcinogenesis in adult female rats, mainly because of different rodent models that are used in chemoprevention studies. The present study was undertaken to compare the tumor-preventative effects of soy protein isolate (SPI) and two of its isoflavones in a "standard" model that had been used for the identification of many chemopreventive agents. Six groups of female Sprague-Dawley rats were provided with modified cornstarch AIN-76A diets supplemented as follows: no additional agents (control), purified genistein (200 mg/kg diet), purified daidzein (200 mg/kg diet), genistein + daidzein (100 mg/kg diet each), SPI containing normal levels of isoflavones (SPI-n), or SPI depleted of isoflavones (SPI-d). Mammary carcinomas were induced by 7,12-dimethylbenz[a]anthracene (DMBA) introduced 1 wk after the animals began consuming the experimental diets. At the end of the study (120 days after DMBA treatment), no significant differences were found among the six groups with respect to tumor incidence or survival, nor was there a significant reduction in tumor multiplicity in the genistein or genistein + daidzein group. However, there was a 32% reduction in tumor multiplicity in the daidzein and SPI-n groups relative to the control group (P < 0.05). The most effective diet was SPI-d, which produced a 50% reduction in tumor multiplicity relative to the control (P < 0.01). The difference between the SPI-d group and the daidzein or SPI-n group was not significant. Median tumor latency was increased from 53 days in the control group to 68 days in the daidzein group and to 72 days in the SPI-d group, but these differences were not statistically significant. These results show that daidzein and SPI (with normal or low levels of isoflavones) are effective inhibitors of DMBA-induced mammary tumors in adult rats.
Analysis of gene expression is dependent on normalization using housekeeping genes. However, many of these housekeeping genes (e.g. GAPDH, beta-actin) are upregulated in chronic pancreatitis and pancreatic cancer, and cannot be used for normalization. For this reason we tried to identify a housekeeping gene useful for expression analysis in pancreatic diseases.
RNA isolated from various tissues and states of disease was subjected to reverse transcription and subsequently amplified by PCR using primers for GAPDH and for the ribosomal highly basic 23-kDa (rb 23-kDa, RPL13A) protein.
As anticipated, expression of GAPDH varied markedly in the different tissues, whereas the expression of rb 23-kDa was constant in all samples investigated.
We recommend the use of the ribosomal highly basic 23-kDa protein as a standard for normalization at least for the pancreas and the prostate.
No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is quite poor. Therefore, we investigated the possible effects of a diet containing GM soybean on mouse exocrine pancreas by means of ultrastructural, morphometrical and immunocytochemical analyses. Our observations demonstrate that, although no structural modification occurs in pancreatic acinar cells of mice fed on GM soybean, quantitative changes of some cellular constituents take place in comparison to control animals. In particular, a diet containing significant amount of GM food seems to influence the zymogen synthesis and processing.
The coat protein (CP) gene of cucumber mosaic virus (CMV) was cloned from a Chinese CMV isolate, the CaMV promoter and NOS terminator added and the gene construct was transformed into both sweet pepper and tomato plants to confer resistance to CMV. Safety assessments of these genetically modified (GM) plants were conducted. It was found that these two GM products showed no genotoxicity either in vitro or in vivo by the micronucleus test, sperm aberration test and Ames test. Animal feeding studies showed no significant differences in growth, body weight gain, food consumption, hematology, blood biochemical indices, organ weights and histopathology between rats or mice of either sex fed with either GM sweet pepper or tomato diets compared with those with non-GM diets. These results demonstrate that the CMV-resistant sweet pepper and tomato are comparable to the non-GM counterparts in terms of food safety.
Three experiments were conducted to compare the feeding value of genetically enhanced corn (Roundup Ready corn events GA21 and nk603) with nontransgenic hybrids. The four treatments included two separate reference hybrids (REF), the near-isogenic control hybrid (CON), and the genetically enhanced corn (RR), resulting in two preplanned comparisons of CON vs. RR and RR vs. the average of REF. In Exp. 1 (RR event GA21), 175 steers (BW = 427 kg) were fed in 25 pens with seven pens per corn hybrid, except CON, which contained four pens due to limited quantities of that hybrid. In Exp. 2 (RR event nk603), 196 steers (BW = 420 kg) were fed in 28 pens with seven pens per corn. In Exp. 3 (RR event nk603), 200 steers were fed in 20 pens, with a similar treatment design to Exp. 2 and five pens per corn. All experiments were conducted as completely randomized designs and utilized corn produced at University of Illinois (Exp. 1 and 2) and University of Nebraska (Exp. 3) research farms under identity-preserved protocols. In all experiments, DMI, ADG, and feed efficiency were similar (P > 0.30) between RR and REF. In Exp. 1 and Exp. 2, RR was not different (P > 0.25) than CON for growth performance. In Exp. 3, RR was not different from CON for ADG and DMI (P > 0.15) or for feed efficiency (P = 0.08). No differences were observed between RR and CON or RR and REF for carcass weight, longissimus dorsi area, and marbling scores in any of the experiments. Subtle differences were observed between RR and either CON or REF for fat depth in each experiment; however, cattle fed RR were not consistently greater and varied from either the CON or the REF (but not both contrasts) within an experiment. Based on these results, insertion of glyphosate-tolerant genes had no significant effect on nutritive quality of corn. Performance and carcass characteristics were not influenced, which suggests that Roundup Ready corn is similar to conventional, nontransgenic corn when fed to finishing feedlot cattle.
In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T0-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T2-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T2-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.
The current study presents the results of a 13 week feeding study in rats with grain from Roundup Ready corn which is tolerant to the herbicide glyphosate. Herbicide tolerance was accomplished through the introduction of cp4 epsps coding sequences into the corn genome for in planta production of CP4 EPSPS enzymes. Unlike related corn EPSPS enzymes, CP4 EPSPS enzymes are not inhibited by the herbicide glyphosate. Purina TestDiets formulated Roundup Ready corn grain into rodent diets at levels of 11 and 33% (w/w). The responses of rats fed diets containing Roundup Ready corn grain were compared to that of rats fed diets containing non-transgenic grain (controls). All diets were nutritionally balanced and conformed to Purina Mills, Inc. specifications for Certified LabDiet 5002. There were 400 rats in the study divided into 10 groups of 20 rats/sex/group. Overall health, body weight, food consumption, clinical pathology parameters (hematology, blood chemistry, urinalysis), organ weights, gross and microscopic appearance of tissues were comparable between groups fed diets containing Roundup Ready and control corn grain. This study complements extensive agronomic, compositional and farm animal feeding studies with Roundup Ready corn grain, confirming it is as safe and nutritious as existing commercial corn hybrids.
This paper aims to examine some of the reasons behind public controversy associated with the introduction of genetically modified foods in Europe the 1990s. The historical background to the controversy is provided to give context. The issue of public acceptance of genetically modified foods, and indeed the emerging biosciences more generally, is considered in the context of risk perceptions and attitudes, public trust in regulatory institutions, scientists, and industry, and the need to develop communication strategies that explicitly include public concerns rather than exclude them. Increased public participation has been promoted as a way of increasing trust in institutional practices associated with the biosciences, although questions still arise as to how to best utilise the outputs of such exercises in policy development. This issue will become more of a priority as decision-making systems become more transparent and open to public scrutiny. The results are discussed in the context of risk assessment and risk management, and recommendations for future research are made. In particular, it is recommended that new methods are developed in order to integrate public values more efficaciously into risk analysis processes, specifically with respect to the biosciences and to technology implementation in general.