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PubMed1992

Nutritional regulation of nucleosomal structure at the chicken malic enzyme promoter in liver.

Transcription of the chicken malic enzyme gene in the liver is stimulated by feeding and inhibited by starvation. Concomitant with the increase in transcription caused by refeeding, chromatin structure around the transcription start site of the malic enzyme gene is modified in the liver. Digestion of chromatin in isolated nuclei with DNase I revealed four feeding-induced DNase I hypersensitive sites (-220, -170, -130 and -70 bp) near the malic enzyme promoter. Similarly, digestion of chromatin with restriction endonucleases detected enhanced cleavage within this region when birds were refed. Micrococcal nuclease detected the presence of nucleosomes over this region in the starved state, but not in the fed state. After food was withdrawn from fed birds, nucleosomes were reformed in this region within 6 h. The speed and magnitude of the changes in nucleosomal structure in this region suggest that they did not require DNA replication.

Animals↗

PubMed2006

Exaggerated response of arginine vasopressin-enhanced green fluorescent protein fusion gene to salt loading without disturbance of body fluid homeostasis in rats.

We examined the effects of chronic salt loading on the hypothalamic expressions of the enhanced green fluorescent protein (eGFP), arginine vasopressin (AVP) and oxytocin (OXT) genes in AVP-eGFP transgenic rats that expressed eGFP in the hypothalamic AVP-containing neurones. In these rats, salt loading for 5 days caused a marked increase of the eGFP fluorescence in the magnocellular divisions of the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the internal layer of the median eminence. Expression of the eGFP gene was increased seven- to eight-fold in the PVN and SON of salt-loaded rats in comparison with euhydrated rats. By contrast, none of these changes were observed in the suprachiasmatic nucleus. The expression of the AVP and OXT genes was increased 1.5- to two-fold in the PVN and SON of salt-loaded nontransgenic (control) and transgenic rats. There were no differences in the expression levels of the AVP and OXT genes in the PVN and SON between nontransgenic (control) and transgenic animals under normal conditions and after salt loading. In the posterior pituitary gland, the intensity of the eGFP fluorescence did not change after salt loading for 5 days, but increased after 10 days of salt loading. Upon salt loading, significant increases in the plasma AVP concentrations, plasma osmolality and plasma Na+ were observed. Furthermore, there were no significant differences in changes of water intake, food intake, urine volume, urine osmolality, urine Na+ concentrations, and the body weights in both models under normal or salt-loaded conditions. Our results show that the response of the AVP-eGFP fusion gene to chronic salt loading is exaggerated, and humoral responses such as AVP and OXT and the body fluid homeostasis are maintained in AVP-eGFP transgenic rats. The AVP-eGFP transgenic rat gives us a new opportunity to study the dynamics of the AVP system in vivo.

Animals↗

PubMed2005

Branched-chain amino acids and brain function.

Branched-chain amino acids (BCAAs) influence brain function by modifying large, neutral amino acid (LNAA) transport at the blood-brain barrier. Transport is shared by several LNAAs, notably the BCAAs and the aromatic amino acids (ArAAs), and is competitive. Consequently, when plasma BCAA concentrations rise, which can occur in response to food ingestion or BCAA administration, or with the onset of certain metabolic diseases (e.g., uncontrolled diabetes), brain BCAA concentrations rise, and ArAA concentrations decline. Such effects occur acutely and chronically. Such reductions in brain ArAA concentrations have functional consequences: biochemically, they reduce the synthesis and the release of neurotransmitters derived from ArAAs, notably serotonin (from tryptophan) and catecholamines (from tyrosine and phenylalanine). The functional effects of such neurochemical changes include altered hormonal function, blood pressure, and affective state. Although the BCAAs thus have biochemical and functional effects in the brain, few attempts have been made to characterize time-course or dose-response relations for such effects. And, no studies have attempted to identify levels of BCAA intake that might produce adverse effects on the brain. The only "model" of very high BCAA exposure is a very rare genetic disorder, maple syrup urine disease, a feature of which is substantial brain dysfunction but that probably cannot serve as a useful model for excessive BCAA intake by normal individuals. Given the known biochemical and functional effects of the BCAAs, it should be a straightforward exercise to design studies to assess dose-response relations for biochemical and functional effects and, in this context, to explore for adverse effect thresholds.

Adolescent↗

PubMed1999

Natural and man-made mutagens and carcinogens in the human diet.

Around 40% of human cancers may relate to dietary factors, including both exogenous and endogenous mutagens. Of exogenous factors, alcohol, certain metals and certain pesticides (both naturally produced or manufactured by the chemical industry), N-nitroso compounds, heterocyclic amines and polycyclic aromatic hydrocarbons are all probable human carcinogens. Despite current negative publicity, genetic engineering appears to be a more precise process and no more likely to lead to cancer risks than conventional breeding processes. Many traditional assessments of cancer hazard from endogenous or exogenous chemicals ignore the presence of modifying factors in the human diet. For example, dietary fat and dietary fibre probably either enhance or protect against cancer, depending upon the exact amounts and chemical nature of the fat fibre. Considerable numbers of other types of antimutagen/anticarcinogens have been identified, with varying modes of action. Additionally, there is an interaction of dietary factors with genetics. Epidemiology will always be important in assessing relative risks, but it is essential to continue developing more sensitive biomonitoring methodologies. It would be desirable to compare precise measures of individual exposure to dietary carcinogens with levels of oxidative damage and evidence of genotoxic effects in a given tissue. Such experimental approaches might be expected to lead a better understanding of the interplay between different dietary factors and also between diet, hereditary and the environment.

Amines↗

PubMed2003

Sustained peripheral expression of transgene adiponectin offsets the development of diet-induced obesity in rats.

Adiponectin (Acrp30) is a physiologically active polypeptide hormone secreted by adipose tissue that shows insulin-sensitizing, antiinflammatory, and antiatherogenic properties. In humans, Acrp30 levels are inversely related to the degree of adiposity. In the current study, we tested the long-term weight-reducing and insulin-enhancing effects of Acrp30 cDNA delivered peripherally by a viral vector. To this end, we have generated a series of recombinant adeno-associated virus vectors of serotypes 1 and 5 encoding mouse Acrp30 cDNAs. The long-term expression of recombinant adeno-associated virus-Acrp30 vectors was tested after intramuscular or intraportal injection in female Sprague-Dawley rats with diet-induced obesity. We show that a single peripheral injection of 10(12) physical particles of Acrp30-encoding vectors resulted in sustained (up to 280 days) significant reduction in body weight, concomitant with the reduction in daily food intake. Acrp30 treatment resulted in higher peripheral insulin sensitivity measured by the i.p. glucose tolerance test in fasted animals. Ectopic expression of the Acrp30 transgene resulted in modulation of hepatic gluconeogenesis and lipogenesis, as demonstrated by the reduction of the expression of two key genes: PEPCK (phosphoenolpyruvate carboxykinase) and SREBP-1c (sterol regulatory element-binding protein 1c) in the liver. These data show successful peripheral therapy in a clinically relevant model for human obesity and insulin resistance.

Adiponectin↗

PubMed2000

Abnormal turning behavior in Drosophila larvae. Identification and molecular analysis of scribbler (sbb).

Our genetic dissection of behavior has isolated scribbler (sbb), a vital gene that encodes a novel protein expressed in the embryonic and larval nervous systems and in the imaginal discs. Larvae with mutations in sbb exhibit abnormally high amounts of turning behavior in the absence of food. sbb is a large gene spanning >50 kb of genomic DNA with four major developmentally regulated transcripts. Transgenic rescue of scribbler behavior was demonstrated by targeting expression of a normal sbb transgene (sbb(+)) expressing one of the major transcripts to the nervous system. The vital function of sbb was restored by ubiquitous expression of this transgene throughout development.

Amino Acid Sequence↗

PubMed2002

Ethical issues for bioscientists in the new millennium.

The scientific understanding of biological processes is developing extremely fast, providing opportunities for changing people's lives in many ways-through health care, food and the environment. The speed with which these changes are occurring means that even bioscientists can only keep up with their own narrow field of science. It is not surprising that members of the public are frightened about the rapidity and impact of the changes arising from the biological revolution. These concerns are often expressed in ethical terms. Decision making about the direction of research and its application is becoming more transparent. This means that bioscientists will have to engage in the debate about their work with members of the public, including those who are opposed to it, in order to create acceptance of their work and its products. At the moment, bioscientists are often ill equipped to enter this debate because of their lack of training in ethics and lack of understanding of the impact of ethics on their work. A better understanding of bioethics will be necessary for entering this debate with vigour. A comprehensive ethical analysis is outside the scope of this text. Some of the principal arguments about the ethics of two aspects of bioscience research-genetically modified crops and the use of experimental animals-will be discussed to illustrate a few of the issues that derive from ethical analyses. I hope that this will encourage toxicologists to take a greater interest in bioethics.

Animal Welfare↗

PubMed2004

Multivitamin production in Lactococcus lactis using metabolic engineering.

The dairy starter bacterium Lactococcus lactis has the potential to synthesize both folate (vitamin B11) and riboflavin (vitamin B2). By directed mutagenesis followed by selection and metabolic engineering we have modified two complicated biosynthetic pathways in L. lactis resulting in simultaneous overproduction of both folate and riboflavin: Following exposure to the riboflavin analogue roseoflavin we have isolated a spontaneous mutant of L. lactis strain NZ9000 that was changed from a riboflavin consumer into a riboflavin producer. This mutant contained a single base change in the regulatory region upstream of the riboflavin biosynthetic genes. By the constitutive overproduction of GTP cyclohydrolase I in this riboflavin-producing strain, the production of folate was increased as well. Novel foods, enriched through fermentation using these multivitamin-producing starters, could compensate the B-vitamin-deficiencies that are common even in highly developed countries and could specifically be used in dietary foods for the large fraction of the Caucasian people (10-15%) with mutations in the methylene tetrahydrofolate reductase (MTHFR).

Folic Acid↗

PubMed2002

Insect feeding mobilizes a unique plant defense protease that disrupts the peritrophic matrix of caterpillars.

Plants frequently respond to herbivorous insect attack by synthesizing defense proteins that deter insect feeding and prevent additional herbivory. Maize (Zea mays L.) lines, resistant to feeding by a number of lepidopteran species, rapidly mobilize a unique 33-kDa cysteine protease in response to caterpillar feeding. The accumulation of the 33-kDa cysteine protease in the maize mid-whorl was correlated with a significant reduction in caterpillar growth that resulted from impaired nutrient utilization. Black Mexican Sweetcorn callus transformed with mir1, the gene encoding the 33-kDa cysteine protease, expressed the protease and growth of caterpillars reared on the transgenic callus was reduced 60-80%. Scanning electron microscopy was used to examine the effect of plant material expressing the 33-kDa cysteine protease on the structure of the caterpillar peritrophic matrix. Because the peritrophic matrix surrounds the food bolus, assists in digestive processes, and protects the caterpillar midgut from physical and chemical damage, disruption of peritrophic matrix may reduce caterpillar growth. The results indicated that the peritrophic matrix was severely damaged when caterpillars fed on resistant maize plants or transgenic Black Mexican Sweetcorn. The accumulation of the 33-kDa cysteine protease in response to caterpillar feeding, and its ability to damage the insect peritrophic matrix, represents an unusual host-plant resistance mechanism that may have applications in agricultural biotechnology.

Animals↗

PubMed1998

Production of the carotenoids lycopene, beta-carotene, and astaxanthin in the food yeast Candida utilis.

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, beta-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promotes and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, beta-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.

Biotechnology↗

PubMed2000

[Endocrine disruption agents: environment, health, public policies, and the precautionary principle].

The already substantial body of evidence and growing web of suspicions as to the scale and severity of the cascade effects of endocrine disrupters (related to persistent organic pollutants or POPs) on the health of ecosystems and humans have sparked such concern that in June 1998, representatives of 94 countries meeting in Montreal under the aegis of UNEP signed a draft international agreement to phase out the most harmful POPs. Related to particular persistent organic pollutants--toxic semi-volatile and persistent chemical compounds now found everywhere in the environment, such as BPCs, organochlorine pesticides, dioxins and furans, that build up in the bodies of organisms that consume other contaminated organisms along the food chain--endocrine disrupters are strongly suspected of affecting the health of animals and adversely impacting the health, fertility and even intellectual faculties of humans. For example, very low-level exposure to some POPs is associated with some hormone-dependent cancers, damage to the central and peripheral nervous systems, impaired immune system function, reproductive disorders and developmental disruptions in newborns and infants, who can be affected in utero or through breast-feeding. Considering the extreme complexity of the scientific and socio-economic effects of POP-related endocrine disrupters, there are those who, advocate a wait-and-see approach, claiming that there is not enough formal scientific proof. There are others who use the available evidence to advance the research, press for bans on incriminated substances and look for global, integrated and viable alternatives. And there are other still who, with careless disregard for the Precautionary Principle, are quite prepared to talk about the perverse effects of POPs in order to justify the increased use of artificial means of reproduction or the replacement of chemical pesticides by pest-resistant genetically modified organisms (GMOs), thereby opening the door to "solutions" that are potentially more biologically and ethically dangerous than the problems they purport to remedy. This paper provides an overview of the current understanding of the main sources and suspected effects of POPs on animal life and human health, explores the complexity of the scientific, economic and political issues involved in any international process to do away with the incriminated products, discusses the risks and perverse consequences of some of the proposed alternatives, and stresses the importance, in the light of these risks and consequences, of placing renewed emphasis on public and environmental health approaches based on the Precautionary Principle.

Endocrine System Diseases↗

PubMed1993

Comparative utilisation of sulphur-containing amino acids by genetically lean or fat chickens.

1. Genetically lean (LL) or fat (FL) male chickens were fed from 28 to 47 days of age on 5 experimental diets differing by their methionine+cystine content (5.4, 5.8, 6.2, 6.6 and 7.0 g/kg, respectively). 2. Growth rate of LL chickens was reduced by the lower sulphur-containing amino acid (SAA) concentrations whereas that of FL was not modified. 3. LL chickens exhibited a larger feather protein gain than FL, which was stimulated by SAA intake. 4. SAA retention, when plotted against SAA consumption, was always greater in LL than in FL. 5. Large differences were observed between genotypes for plasma-free amino acids. Lysine, glutamic acid, histidine and serine were found at significantly higher concentrations in LL birds. Branched amino acids, aromatic amino acids, SAA and arginine were found at higher concentrations in FL. No differences were observed for aspartic acid, glycine, alanine and total amino acids. Methionine supplementation decreased free amino acid concentrations, with the exceptions of arginine and leucine. 6. It is concluded that lean chickens require a higher dietary concentration of SAA than FL. This is mainly caused by their lower food consumption and their greater feather synthesis. However, LL use SAA more efficiently than FL.

Amino Acids↗

PubMed2003

Is the food-entrainable circadian oscillator in the digestive system?

Food-anticipatory activity (FAA) is the increase in locomotion and core body temperature that precedes a daily scheduled meal. It is driven by a circadian oscillator but is independent of the suprachiasmatic nuclei. Recent results that reveal meal-entrained clock gene expression in rat and mouse peripheral organs raise the intriguing possibility that the digestive system is the site of the feeding-entrained oscillator (FEO) that underlies FAA. We tested this possibility by comparing FAA and Per1 rhythmicity in the digestive system of the Per1-luciferase transgenic rat. First, rats were entrained to daytime restricted feeding (RF, 10 days), then fed ad libitum (AL, 10 days), then food deprived (FD, 2 days). As expected FAA was evident during RF and disappeared during subsequent AL feeding, but returned at the correct phase during deprivation. The phase of Per1 in liver, stomach and colon shifted from a nocturnal to a diurnal peak during RF, but shifted back to nocturnal phase during the subsequent AL and remained nocturnal during food deprivation periods. Second, rats were entrained to two daily meals at zeitgeber time (ZT) 0400 and ZT 1600. FAA to both meals emerged after about 10days of dual RF. However, all tissues studied (all five liver lobes, esophagus, antral stomach, body of stomach, colon) showed entrainment consistent with only the night-time meal. These two results are inconsistent with the hypothesis that FAA arises as an output of rhythms in the gastrointestinal (GI) system. The results also highlight an interesting diversity among peripheral oscillators in their ability to entrain to meals and the direction of the phase shift after RF ends.

Animals↗

PubMedDate not supplied

Mating propensities and variations in enzyme activities in long-term cage populations of Drosophila melanogaster.

Environment-dependent reproductive isolation was established between cage populations (Bs) of Drosophila melanogaster originated from a Greek natural population (summer 1973) and maintained for about five years under different diets (poor-rich). The detected deviation from random mating involved no homogametic or heterogametic preference but rather, a significantly increased activity of males from populations maintained on the rich food medium. This observation indicates that the male parental investment is not negligible and under certain conditions sexual isolation can be a function not only of female behavior but also of male behavior. Differences also were found in various enzyme activities on the inter- and intra-population levels. Given those observations as well as the observed different behavioral patterns of Bs and Cs-Ds populations 19, a preliminary attempt was made to associate adaptive evolution with differences in enzyme activities. The differences in enzyme activities between populations reared on different media are not due to allozymic differences. It also was shown that in some populations environmental effects do not always elicit differences in enzyme activity. It was concluded, therefore, that the observed variations were the result of environmental effects interacting with modifier genes.

Animals↗

PubMed1985

Nutritional influences on dietary selection patterns of obese and lean Zucker rats.

Little is known about the behavioral, nutritional, and metabolic events that control dietary intake quality. Two experiments are described that manipulate nutritional conditions that have been hypothesized to affect dietary item choice so as to assess what effect, if any, the added factor of genetic obesity has in modifying the response to these manipulations. In the first experiment, 5 week old male obese and lean Zucker rats were fed a diet that varied in protein content (10%, 20%, or 60% casein by weight) for ten weeks. They were then allowed to select a diet from three separate macronutrient sources (casein, starch, or corn oil). Although body weights at the end of the 10 week maintenance period were markedly different, selection patterns were not influenced by pre-feeding different levels of protein. Obese rats selected a diet that was higher in fat and lower in protein than the diets composed by lean rats. In the second experiment, four groups of 7 month old obese and lean Zucker rats were given access to one of four diets that varied in protein content (5%, 10%, 15%, or 20% casein by weight). In addition, each rat was periodically given access to a 32% sucrose solution. Access to sucrose promoted increases in total caloric intake, independent of the protein content of the diet. Obese rats typically ate more calories per day than did their lean littermates. Results from these experiments suggest that food item selection is determined more by factors associated with obesity than by factors associated with dietary history.

Animal Nutritional Physiological Phenomena↗

PubMed2000

Growth efficiency in transgenic tilapia (Oreochromis sp.) carrying a single copy of an homologous cDNA growth hormone.

Growth hormone (GH) has been shown to have a profound impact on fish physiology and metabolism. However, detailed studies in transgenic fish have not been conducted. We have characterized the food conversion efficiency, protein profile, and biochemical correlates of growth rate in transgenic tilapia expressing the tilapia GH cDNA under the control of human cytomegalovirus regulatory sequences. Transgenic tilapia exhibited about 3.6-fold less food consumption than nontransgenic controls (P < 0.001). The food conversion efficiency was significantly (P < 0.05) higher (290%) in transgenic tilapia (2.3 +/- 0.4) than in the control group (0.8 +/- 0.2). Efficiency of growth, synthesis retention, anabolic stimulation, and average protein synthesis were higher in transgenic than in nontransgenic tilapia. Distinctive metabolic differences were found in transgenic juvenile tilapia. We had found differences in hepatic glucose, and in agreement with previous results we observed differences in the level of enzymatic activities in target organs. We conclude that GH-transgenic juvenile tilapia show altered physiological and metabolic conditions and are biologically more efficient.

Animals↗

PubMed1999

Use of plant roots for phytoremediation and molecular farming.

Alternative agriculture, which expands the uses of plants well beyond food and fiber, is beginning to change plant biology. Two plant-based biotechnologies were recently developed that take advantage of the ability of plant roots to absorb or secrete various substances. They are (i) phytoextraction, the use of plants to remove pollutants from the environment and (ii) rhizosecretion, a subset of molecular farming, designed to produce and secrete valuable natural products and recombinant proteins from roots. Here we discuss recent advances in these technologies and assess their potential in soil remediation, drug discovery, and molecular farming.

Agriculture↗

PubMed2005

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes.

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.

Amines↗