Integrated histopathological and urinary metabonomic investigation of the pathogenesis of microcystin-LR toxicosis.

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium.

Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained by the transformed cell lines.

Heat stress affects blood-brain barrier permeability to horseradish peroxidase in mice.

The blood-brain barrier of mice subjected to hyperthermia for 0-135 min was examined using horseradish peroxidase (HRP) and Evans blue dye tracers by light, fluorescence and electron microscopy. Neither control nor heat-stressed mice exhibited extravasation of the Evans blue dye-albumin complex from the brain microvasculature. Gross examination of vibratome sections processed to reveal HRP reaction product exhibited multiple microfoci of HRP extravasation. Electron microscopic observations of the heat-stressed tissues revealed HRP reaction product within pinocytotic vesicles, tubulo-vesicular complexes, transendothelial channels and occasionally flooded within the cytoplasm of endothelial cells. HRP reaction product could clearly be seen in the basal laminae of the capillaries and in the surrounding neuropil. This study demonstrates that blood-brain barrier permeability to HRP is increased in response to heat stress and introduces a new, reproducible model of blood-brain barrier disruption.

Food additives and food components in total diets in the Netherlands.

1. During a period of 2 years, every 2 months 126 different food items forming a 'market basket' were purchased, prepared and divided into twelve food commodity groups. The 'market basket' was based on a study of the dietary pattern of 16- to 18-year-old male adolescents. In the (homogenized) food groups various additives and components of nutritional importance were determined. From the concentrations of the additives and components in the food groups and the daily consumption of each food group, a mean daily intake of all components analysed was calculated. 2. The mean daily amounts of benzoic acid (34 mg), sorbic acid (6 mg), glutamic acid (660 mg) and sulphite (3 mg) were all far below the acceptable daily intake (ADI) value. Butylated hydroxytoluene and gallates were not detectable, while butylated hydroxyanisole (BHA) was found in only a few instances; the maximum amount of BHA was also very low (4 mg). 3. The mean daily intakes of fluorine (0.8 mg), iodine (0.21 mg), phosphorus (1860 mg) and alpha-tocopherol (9.4 mg) seem safe and adequate. Cholesterol intakes of 25% above the maximum of 300 mg/d, as advised by the Dutch Bureau for Nutrition Education, were found. The mean fat intake appeared to be 40% of total daily energy, protein content 13% of total energy and total (available) carbohydrate 46% of total energy. The daily dietary fibre content (18 g) and the daily amount of linoleic + linolenic acid (6% of total energy) were considered too low. The daily level of sodium (4.2 g) was not considered too high. 4. It is recommended that the study should be repeated regularly, e.g. every few years, in order to monitor trends in the concentrations of significant food components in total diets.

[On the iron contamination in cocoa and chocolate products (author's transl)]. / Zur Eisenkontamination in Kakao und Kabaoerzeugnissen.

The investigation in question deals with the iron contamination in cocoa and chocolate products. Semi finished goods as well as finished products were examined. An average of 29 mg/kg total iron (i.e. ionic + metallic iron) was found in cocoabeans. The iron content of cocoa shells was approximately 10 fold higher. The process of grinding roasted nibs to cocoa-mass (liquor) resulted in a noticeable increase of the quantity of iron to an average amount of 150 mg/kg mass. This process thus produced an increase of the iron content of approximately 75 to as much as 200%. The cocoa powders contained more iron (238 mg/kg) than the cocoa mass, which linked to a reduction of the fat content by pressing and by grinding of the presscake. The quantities of iron observed in commercial samples of cocoa powder from different countries did not show appreciable differences.