Inhibition of acrylamide toxicity in mice by three dietary constituents

The inhibitory effects of three dietary constituents, tea polyphenols, resveratrol, and diallyl trisulfide, on acrylamide-biomacromolecule (liver DNA, protamine, and hemoglobin) adduct formation at human exposure level were studied by accelerator mass spectrometry. The results demonstrated that the three dietary constituents all significantly inhibited the formation of acrylamide adducts with liver DNA, whereas tea polyphenols and diallyl trisulfide reduced protamine and hemoglobin adducts, respectively. Further biochemical studies showed that acrylamide could significantly inactivate creatine kinase and glutathione S-transferase and deplete glutathione. When the inhibitors were cotreated with acrylamide, all of them could effectively recover the activities of creatine kinase. In addition, tea polyphenols and diallyl trisulfide could increase glutathione S-transferase activity remarkably. On the basis of these results, mechanisms of the effects are discussed. This study might provide a beneficial guide to people's diet for the purpose of reducing the harmful effect of acrylamide.     

Review of methods for the reduction of dietary content and toxicity of acrylamide

Potentially toxic acrylamide is largely derived from heat-induced reactions between the amino group of the free amino acid asparagine and carbonyl groups of glucose and fructose in cereals, potatoes, and other plant-derived foods. This overview surveys and consolidates the following dietary aspects of acrylamide: distribution in food originating from different sources; consumption by diverse populations; reduction of the acrylamide content in the diet; and suppression of adverse effects in vivo. Methods to reduce adverse effects of dietary acrylamide include (a) selecting potato, cereal, and other plant varieties for dietary use that contain low levels of the acrylamide precursors, namely, asparagine and glucose; (b) removing precursors before processing; (c) using the enzyme asparaginase to hydrolyze asparagine to aspartic acid; (d) selecting processing conditions (pH, temperature, time, processing and storage atmosphere) that minimize acrylamide formation; (e) adding food ingredients (acidulants, amino acids, antioxidants, nonreducing carbohydrates, chitosan, garlic compounds, protein hydrolysates, proteins, metal salts) that have been reported to prevent acrylamide formation; (f) removing/trapping acrylamide after it is formed with the aid of chromatography, evaporation, polymerization, or reaction with other food ingredients; and (g) reducing in vivo toxicity. Research needs are suggested that may further facilitate reducing the acrylamide burden of the diet. Researchers are challenged to (a) apply the available methods and to minimize the acrylamide content of the diet without adversely affecting the nutritional quality, safety, and sensory attributes, including color and flavor, while maintaining consumer acceptance; and (b) educate commercial and home food processors and the public about available approaches to mitigating undesirable effects of dietary acrylamide.     

Literature review on higher plants for toxicity testing

Phytotoxicity tests using higher plants in general are infrequently used as a part of ecotoxicology. Many reports assess herbicide toxicity merely on the basis of faunal species tests. This is inadequate because the herbicide impact is much greater on flora than on fauna. Environmental pollution by herbicides was likely to have been quite wide-spread during the past years (1964–1984) when the use of herbicides grew five-fold. When herbicides reach non-target areas, they can cause unacceptable harm to non-target species, plants in particular. The toxicity of herbicides to algal species is not likely to be identical to that of higher plants, so that algal species may not serve as a surrogate species for the toxicity evaluation. Currently there are two promising phytotoxicity tests. Common duckweed is an aquatic species and sensitive to toxicity. Duckweek test can be used with static, renewal, or flow-through methods. The latter two are especially useful for unstable compounds or samples. Seed germination and root elongation tests are versatile and can be tested in water, wastewater, sediment, and slurry. Many recent activities in these areas suggest that phytotoxicity tests are a valuable part of ecotoxicology.     

Using dietary exposure and physiologically based pharmacokinetic/pharmacodynamic modeling in human risk extrapolations for acrylamide toxicity

The discovery of acrylamide (AA) in many common cooked starchy foods has presented significant challenges to toxicologists, food scientists, and national regulatory and public health organizations because of the potential for producing neurotoxicity and cancer. This paper reviews some of the underlying experimental bases for AA toxicity and earlier risk assessments. Then, dietary exposure modeling is used to estimate probable AA intake in the U.S. population, and physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling is used to integrate the findings of rodent neurotoxicity and cancer into estimates of risks from human AA exposure through the diet. The goal of these modeling techniques is to reduce the uncertainty inherent in extrapolating toxicological findings across species and dose by comparing common exposure biomarkers. PBPK/PD modeling estimated population-based lifetime excess cancer risks from average AA consumption in the diet in the range of 1-4 x 10 (-4); however, modeling did not support a link between dietary AA exposure and human neurotoxicity because marginal exposure ratios were 50-300 lower than in rodents. In addition, dietary exposure modeling suggests that because AA is found in so many common foods, even big changes in concentration for single foods or groups of foods would probably have a small impact on overall population-based intake and risk. These results suggest that a more holistic analysis of dietary cancer risks may be appropriate, by which potential risks from AA should be considered in conjunction with other risks and benefits from foods.     

Appraisal of existing methodology in aspiration toxicity testing.

New methods or modifications of currently used methods in aspiration toxicology must be capable of accurately assessing the hazard and toxicity potentials of those products which are capable of being aspirated. The methods evaluated in the rat were intratracheal injections,intravenous injections, and modifications of the Gerarde technique. A product containing petroleum distillates and a standard viscosity petroleum distillate sample were administered to Osborne-Mendel rats, rabbits, and guinea pigs in ml/kg doses. The utility of the methods was determined by comparing lung weight increases, lung-body weight ratios, gross pathological lung changes, and mortality. The modified Gerarde technique showed the best potential for predicting aspiration hazard and toxicity based on the severity of the reactions which, using a radiolabeled oil, was shown to be a function of the amount of product which enters and reacts with the lungs. The influence of preventing the swallowing reflex to assure aspiration of the oil was assessed in the rat. The results show that less than 10% of the dose is aspirated when the swallowing reflex occurs. Methods of sacrifice were investigated for an effect on rat lung weight. No tested method except stunning with cervical dislocation produced an effect. Tests were conducted on rats and rabbits to determine differences in lung reactivity to the influence of inhalation anesthetics, pentothal sodium, or no anesthesia in response to petroleum distillate administration and to determine which test species is the better animal model. The resuls indicate that the rat is the better test model because of degree of lung reaction, size, and economy, and that inhalation anesthesia with ether is preferable over the other choices.     

Development of an alternative artificial soil for earthworm toxicity testing in tropical countries

The standard soil invertebrate toxicity tests developed by OECD and ISO use an artificial soil as the test substrate, which contains sphagnum peat as a component. This type of peat is not widely available. Investigation of possible alternative substrates using locally available materials therefore is vital for performing such ecotoxicity tests, particularly in the tropics. We studied the suitability of paddy husk (PH), saw-dust (SD), non-composted (NCCP) and composted coco peat (CCP) as a replacement for sphagnum peat. Artificial soil (AS) was prepared by mixing 70% sand and 20% kaolin clay with 10% PH, SD, NCCP or CCP. First, the reproduction potential of the earthworm Eisenia andrei was investigated in modified artificial soil (MAS) using the original OECD AS as the control. The number of juveniles produced in OECD AS, MAS_PH and MAS_CCP was not significantly different but it was significantly reduced (p < 0.05) in MAS_SD and MAS_NCCP. The toxicity of chlorpyrifos, carbendazim and carbofuran for E. andrei was determined to validate the substrates. The 28-day LC₅₀s for the three pesticides in original AS, MAS_CCP and MAS_PH were not significantly different, but the EC₅₀ for effects on reproduction in the MAS_PH was significantly lower (p < 0.05) compared to OECD AS and MAS_CCP. We conclude that composted coco peat might be a suitable replacement for sphagnum peat in AS for soil ecotoxicity studies.     

Original Papers: Manganese toxicity of melon plants growing on an isolated soil bed after steam sterilization

(Jpn. J. Soil Sci.Plant Nutr., 77, 257–263, 2006)

The symptom of blighted leaves was observed for the melon plants grown on an isolated soil bed after steam sterilization (SS) in a greenhouse in Kochi Prefecture, Japan. To clarify the causes of this symptom, soil and plant analyses were conducted in the first experiment. Manganese concentrations in the leaves with the symptom were higher than 1,000 mg kg?1and exchangeable Mn in the soil was higher than 30 mg kg?1, which indicated the observed symptom was due to Mn excess. In the second experiment, formation of Mn oxide-dissolving substances after SS was examined for several organic amendments, because large amounts of palm chips had been supplied to the soil before SS in the first experiment. As a treatment similar to SS, palm chips, bark compost, and cattle feces manure were autoclaved. The water extracts were collected before and after the autoclave treatment and the Mn oxide-dissolving capacities of the extracts were evaluated. The Mn oxide-dissolving capacity of the water extracts after SS was remarkably higher than before SS, and the value was highest in palm chips, and followed by bark compost, and cattle feces manure. Fractionation and HPLC analysis of the water extracts after SS revealed that the main substance behind Mn oxide reduction in palm tips was arabinose and that in bark manure was malic acid. Further, Mn oxide-dissolving capacities of the water extracts were almost completely explained by the amounts of arabinose or malic acid found in these materials. From these results, it was considered that reducing substances such as arabinose were released by SS from the palm chips which had been applied in large quantity before SS and these reducing substances dissolved the soil Mn oxides and increased the amounts of available Mn, which led to the occurrence of Mn toxicity to the plants.     

Optimization of chick embryotoxicity bioassay for testing toxicity potential of fungal metabolites

The optimization of a simple, sensitive procedure using a chick embryotoxicity screening test (CHEST) bioassay for detection of toxic compounds is presented. Dosing protocols of eggs, using several mycotoxins (aflatoxin B1, deoxynivalenol, T-2 toxin) and appropriate controls, were evaluated for embryonic sensitivity, overall practicality of the procedure, and consistency of results. It was found that both type of carrier solvent and volume injected could significantly affect overall embryonic mortality. The chick embryo was most sensitive to the effects of toxins and solvents after 1 or 2 days of incubation; a rapid decrease in response was observed as the age of the embryo at dosing increased. Following administration of the toxins just below the shell membrane by way of a small hole (less than 0.5 mm diameter) punched in the shell, a good dose-response (% mortality) could be obtained regardless of the site of injection (except directly into the yolk), although dosing via the air sac position resulted in a slightly better statistical outcome. Although some variations in calculated LD50 values were found among repeated assays, statistical analyses showed that the differences were not due to dosing protocol but to the variations in embryo sensitivities among batches of eggs. Thus, if standard reference toxins for comparison are run concurrently, the CHEST assay can prove to be a very satisfactory model, as well as having considerable flexibility to be adapted to the needs and resources of many laboratories.     

Field application of industrial by-products as Al toxicity amendments: chemical and mineralogical implications

Lime, gypsum and various gypsum‐like by‐products have long been applied to soil surfaces as ameliorants of soil acidity and aluminium and manganese toxicity. We examined changes in chemical and mineralogical properties at two different depths in two acid soils one year after the application of gypsum, phosphogypsum + dolomitic residue, red gypsum + dolomitic residue, sugar foam, and sugar foam + mined gypsum. All treatments were found to increase the proportion of Ca²⁺ and decrease those of Al³⁺ and Mn²⁺ in the exchange complex of the surface and subsurface horizons, thus reducing its effective Al and Mn saturation. However, the mined gypsum treatment resulted in losses of Mg²⁺ from the Ap horizon of the soils, and the sugar foam treatment was not so effective with the AB horizons as the other treatments. The combined application of both gypsum‐like by‐products and the dolomitic residue proved the most effective choice with a view to reducing the effective Al and Mn saturation of the exchange complex in the Ap and AB horizons. In addition, both treatments reduced Mg²⁺ losses at both depths. Finally, all treatments resulted in the formation and retention on mineral and organic surfaces of a large fraction of the Al³⁺ released by exchange with Ca²⁺ as Al polymers. This is quite consistent with the observed changes in the CuCl₂‐, oxalate‐ and DTPA‐extractable Al contents as well as by SEM and EDS analyses. Based on these results, the use of the appropriate mixtures of these by‐products is an effective alternative to that of mined gypsum and lime to alleviate soil acidity and reduce toxic concentrations of Al³⁺ and Mn²⁺ in agricultural acid soils.     

Effectiveness of methods for reducing acrylamide in bakery products

Pilot-scale bread, biscuit, and cracker doughs have been baked to assess how well recipe changes could reduce acrylamide in commercial bakery products. Removing ammonium-based raising agents was beneficial in biscuits. In doughs, long yeast fermentations were an effective way of reducing asparagine levels and hence acrylamide. At moderate fermentation times fructose levels increased, but the yeast later absorbed this, so the net effect on acrylamide was beneficial. Metal ions such as calcium reduced acrylamide when added as the carbonate or chloride. Hence, the fortification of flour with calcium carbonate, over and above its natural mineral content, has an additional benefit. However, some other possible methods of adding calcium to bakery doughs, for example, via the permitted preservative calcium propionate, were not beneficial. Amino acid addition to doughs gave modest reductions in acrylamide. Lowering the dough pH reduced acrylamide, but at the expense of higher levels of other process contaminants such as 3-monochloropropane-1,2-diol (3-MCPD).     

Rugged LC-MS/MS survey analysis for acrylamide in foods

The described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of acrylamide in food entails aqueous room temperature extraction, SPE cleanup, and analysis by LC-MS/MS. The method is applicable to a wide variety of foods. [(13)C(3)]acrylamide is the internal standard. The limit of quantitation is 10 ppb (microg/kg). Data were obtained in duplicate from >450 products representing >35 different food types. The variability in analyte levels in certain food types suggests that it may be possible to reduce acrylamide levels in those foods.     

Soil ciliate bioassay for the pore water habitat a missing link between microflora and earthworm testing in soil toxicity assessment

Background, Scope and Goal. The chemical, pesticide and biocide legislation of the European Union assembles a variety of bioassays. Among the ecotoxicological tests involved, the testing strategy for the aquatic compartment builds up on three tests reflecting the main trophic levels (algae,Daphnia, fish). For the soil compartment at least one trophic level for a basic food chain is missing, namely between microflora and earthworms. Protozoa are an ideal missing link as they were shown to be the most prominent faunal contributors to nutrient cycling in soil ecosystems and as they represent the lacking first level consumers as well as the highly diverse microfauna. As protozoa inhabit the soil pore water, they can serve as direct indicators for the solved and thus bioavailable portion of xenobiotics. In order to widen the spectrum of available toxicity tests for a meaningful effect assessment for the soil compartment, a test with the soil ciliateColpoda inflata (Ciliophora, Protozoa), introduced by Pratt et al. (1997), was improved.     

Toward a kinetic model for acrylamide formation in a glucose-asparagine reaction system

A kinetic model for the formation of acrylamide in a glucose-asparagine reaction system is pro-posed. Equimolar solutions (0.2 M) of glucose and asparagine were heated at different temperatures (120-200 degrees C) at pH 6.8. Besides the reactants, acrylamide, fructose, and melanoidins were quantified after predetermined heating times (0-45 min). Multiresponse modeling by use of nonlinear regression with the determinant criterion was used to estimate model parameters. The proposed model resulted in a reasonable estimation for the formation of acrylamide in an aqueous model system, although the behavior of glucose, fructose, and asparagine was slightly underestimated. The formation of acrylamide reached its maximum when the concentration of sugars was reduced to about 0. This supported previous research, showing that a carbonyl source is needed for the formation of acrylamide from asparagine. Furthermore, it is observed that acrylamide is an intermediate of the Maillard reaction rather than an end product, which implies that it is also subject to a degradation reaction.     

Protein/energy ratios of current diets in developed and developing countries compared with a safe protein/energy ratio: implications for recommended protein and amino acid intakes

Revised estimates of protein and amino acid requirements are under discussion by the Food and Agriculture Organization (FAO)/World Health Organizaion (WHO), and have been proposed in a recent report on Dietary Reference Intakes (DRIs) from the USA. The nature and magnitude of these requirements are not entirely resolved, and no consideration has been given to the potential influence of metabolic adaptation on dietary requirements. We have examined the implications of these new values, and of the conceptual metabolic framework in which they are used, for defining the nutritional adequacy of protein intakes in developed and developing countries. We have expressed proposed values for protein requirements in relation to energy requirements, predicted for physical activity levels of 1.5, 1.75 and 2.0 times basal metabolic rate, in order to generate reference ratios for protein energy/total energy (reference P/E ratio) as a function of age, body weight, gender and physical activity level. Proposed values for amino acid requirements have been used to adjust the available digestible P/E ratio of foods and diets for protein quality. Focusing on the diets of UK omnivores and vegetarians and on diets in India, the risk of protein deficiency is evaluated from a comparison of P/E ratios of metabolic requirements with protein-quality-adjusted P/E ratios of intakes. A qualitative and conservative estimate of risk of deficiency is made by comparing the adjusted P/E ratio of the intake with a reference P/E ratio calculated for age, body weight, gender and physical activity according to FAO/WHO/United Nations University. A semi-quantitative estimate of risk of deficiency has also been made by the cut point approach, calculated as the proportion of the intake distribution below the mean P/E ratio of the requirement. Values for the quality-adjusted P/E ratio of the diet range from 0.126 for the UK omnivore diet to 0.054 for a rice-based diet of adults in West Bengal, which is lysine-limited, falling to 0.050 for 1-year-old children. The reference P/E ratio for men and women increases with age, is higher for females than males, is higher for small compared with large adults at any age and decreases with physical activity. Thus if a particular diet is potentially limiting in protein, protein deficiency is most likely in large, elderly sedentary women followed by the adolescent female and least likely in moderately active young children, the opposite of what has usually been assumed. Within the currently accepted framework, the diets do not meet the protein needs of the entire population of the UK, have a significant risk of deficiency throughout India for all except extremely active small adults, and are grossly inadequate for all population groups, apart from physically active young children in West Bengal, regardless of body weight or level of food intake. The lysine limitation of the cereal-based Indian diets is dependent on the choice of lysine requirement values from the published range. We consider that the value selected is too high, because of uncertainties and inconsistencies in the approaches used. A more appropriate choice from the lower end of the range would remove the lysine limitation of cereal-based diets, and reduce some of the perceived risk of deficiency. However, diets remain limited by the amount of digestible protein for many population groups, especially in West Bengal. In the context of risk management, one option would be to accept the current values and the conceptual metabolic framework within which they have been derived. This would have major implications for the supplies of high-quality protein to the developing countries. An alternative option would be to re-evaluate the currently proposed values for the requirements for protein and amino acids. We conclude that the choice of values for the adult lysine requirement should be re-evaluated and that serious consideration should be given to the extent to which adaptive mechanisms might enable the metabolic requirement for protein to be met from current intakes. This will entail a better understanding of the relationships between dietary protein and health.     

Technetium-99 toxicity in phaseolus vulgaris: Ultrastructural evidence for metabolic disorders

The influence of light intensity on the effects of 10^–6 mol L^{–1 99}Tc on growth, chlorophyll and carotenoid contents of bush bean plants was investigated. After germination and cotyledon excision, the plants were grown in a growth chamber either under low light (photosynthetic active radiation, PAR 144 E m^–2 s^–1) or higher light (PAR 307 E M^–2 s^–1) conditions. In plants grown under the higher light conditions, ⁹⁹Tc hardly affected CO₂-assimilation, dark respiration, pigment contents and growth. No toxicity symptoms were observed in these plants. Under low light conditions, ⁹⁹T c significantly decreased growth and the concentration of chlorophylls. Toxicity symptoms in the form of chlorosis and necrosis developed. Transmission electron microscopy (TEM) observations revealed alterations of chloroplast ultrastructure comparable to those described for plants slightly affected by paraquat toxicity or by Mo-deficiency. Our results indicate that in the low light plants ⁹⁹Tc induces damage in chloroplasts by peroxidation of membrane lipids.     

Habitat associations of bats in Northern Ireland: implications for conservation

Agricultural intensification in Northern Ireland has brought about large-scale changes to the landscape with a detrimental effect on biodiversity. Between 1996 and 1998, we surveyed a stratified random sample of 1 km squares for bats using a spot-sample technique and time expansion bat detector to establish linear and area habitat associations. Bats strongly selected water bodies with bankside vegetation, treelines, and deciduous and mixed woodland edge, avoiding open areas such as upland/unimproved grassland and improved grassland. Of three sympatric pipistrelle species (Pipistrellus pipistrellus, P. pygmaeus and P. nathusii), two were shown to forage in different habitats; P. pipistrellus being more of a ‘generalist’, foraging in a wider variety of habitats than P. pygmaeus, a ‘specialist’ which foraged in a smaller range of habitats. Nyctalus leisleri selected parkland/amenity grassland, deciduous woodland edge and rivers/canals and avoided improved grassland, hedgerows, whilst Myotis spp. selected deciduous woodland centre, lake/reservoir margins and rivers/canals. Reduction in area and quality of inland water, deciduous woodlands and field boundaries in Northern Ireland may impact on bat populations. Habitat management should focus on improving those habitats which are selected by bats, including the maintenance and enhancement of connecting linear habitats.     

Tests for the depolymerization of polyacrylamides as a potential source of acrylamide in heated foods

This study was conducted to test the possibility that polyacrylamides used in agriculture may contribute to acrylamide formation in heated foods by thermal depolymerization. Two samples of polyacrylamide with low molecular weights of 1.5 and 10 kDa were used to test for in-chain and end-chain depolymerization. They were added as aqueous solutions to filter paper for heating in a drying/dry environment and added to a cooking oil for heating in a fatty environment. The heating conditions were 175 degrees C for 15 and 30 min, respectively. Both regimens were tested in the absence and presence of the redox-active metal ions Fe(III) and Cu(II), and all tests were conducted without the exclusion of atmospheric oxygen. There was no evidence of any significant depolymerization of polyacrylamide to free acrylamide monomer, <0.04%. In fact, residual levels of acrylamide present already in the low molecular weight polymers were seen to fall by 50-80% on heating. Consequently, it is concluded that even if polyacrylamides were to contaminate agricultural crops and foods derived therefrom (which itself is an unproven suggestion), there is no evidence that the polymers would depolymerize on heating of the food to form acrylamide in any significant amount.     

Conspecific attraction in loggerhead shrikes: implications for habitat conservation and reintroduction

The loggerhead shrike, Lanius ludovicianus, is a declining songbird that forms breeding aggregations. Despite such reports from several populations, only one statistical analysis of loggerhead shrike territory distribution has been published to date. I use a spatio-temporal simulation technique to test for deviations from randomness in the spatial distribution of point data that takes into account date of nest establishment. I apply this model to data on the distribution of shrike nests in Comanche County, Oklahoma, USA, collected over the 1998 and 1999 breeding seasons. When the data are considered without regard to date of nest-establishment, the results are equivocal; nests are significantly aggregated in 1999, but not 1998. However, when order of nest establishment is taken into account, later nests are significantly more likely to be closer than expected to conspecifics in both years. This is true even when the distribution of simple resources, such as suitable nest-trees, is included in the model. These results are consistent with the hypothesis that loggerhead shrikes ‘prospect’ for suitable habitat using the distribution of breeding conspecifics. This aspect of loggerhead shrike breeding ecology should be considered for both habitat conservation and captive breeding and reintroduction programs for this species.