Toxicological potential of 2-alkylcyclobutanones--specific radiolytic products in irradiated fat-containing food--in bacteria and human cell lines.

Food irradiation has been considered as a safe processing technology to improve food safety and preservation, eliminating efficiently bacterial pathogens, parasites and insects. This study aims to characterize the toxicological potential of 2-alkylcyclobutanones (2-ACBs), radiolytic derivatives of triglycerides, formed uniquely upon irradiation of fat-containing food. In irradiated food they are generated proportionally to fat content and absorbed radiation dose. The cyto- and genotoxic potentials of various highly pure synthetic 2-ACBs were studied in bacteria and human cell lines. While pronounced cytotoxicity was evident in bacteria, no mutagenic activity has been revealed by the Ames test in Salmonella strains TA 97, TA 98 and TA 100. In mammalian cells genotoxicity was demonstrated mainly by the induction of DNA base lesions recognized by the Fpg protein as determined by both the Comet Assay and the Alkaline Unwinding procedure. Formation of DNA strand breaks was observed by the Alkaline Unwinding procedure but not by the Comet Assay. The extent of cytotoxicity and genotoxicity were dependent on chain length and degree of unsaturation of the fatty acid chain. Further studies will have to clarify mechanisms of action and potential relevance for human exposure situation.

Use of the DNA Comet Assay to detect beef meat treated by ionizing radiation.

The DNA Comet Assay has been described as a rapid and inexpensive screening test to identify radiation treatment of food. In this work, this method was applied to detect the treatment of beef meat pieces either by gamma rays or electron beam. The dose levels were 2.5, 4.5, and 7.0kGy for chilled samples, and 2.5, 4.5, 7.0 and 8.5kGy for frozen samples. The analyses were made over periods of 15 and 30 days after irradiation for the chilled and frozen samples, respectively. The effects of gamma rays and electron beam on DNA migration in the test were similar. The DNA Comet Assay, under neutral conditions, made it easy to discriminate between irradiated and non-irradiated beef.

Detection of 2-alkylcyclobutanones, markers for irradiated foods, in adipose tissues of animals fed with these substances.

Laboratory rats received a freshly prepared drinking fluid containing 0.005% 2-tetradecyl- or 2-tetradecenyl-cyclobutanones daily for 4 months. These two compounds were recovered in the adipose tissues of the animals that consumed them. Less than 1% of the 2-alkylcyclobutanones ingested daily were excreted in the feces. In addition, our data indicate that 2-alkylcyclobutanones are able to cross the intestinal barrier, to enter into the bloodstream, and to be stored in the adipose tissue of an animal. However, the amounts of these substances detected in the adipose tissues and in the feces were much smaller than the amounts ingested.

Moderate intervention with carotenoid-rich vegetable products reduces lipid peroxidation in men.

Because of their antioxidant properties, carotenoids may have beneficial effects in preventing cancer and cardiovascular disease. However, in humans consuming carotenoid-rich vegetables, data concerning the antioxidant effects of carotenoids are rather scarce. A human intervention trial was conducted, therefore, to determine whether a moderately increased consumption of carotenoid-rich vegetables would influence the antioxidant status in 23 healthy men. This short-term feeding study lasted 8 wk during which the men consumed a low carotenoid diet. A 2-wk low carotenoid period was followed by daily consumption of 330 mL tomato juice, then by 330 mL carrot juice and then by 10 g of spinach powder, each for 2 wk. Antioxidant status [water-soluble antioxidants in serum, ferric reducing ability of plasma (FRAP) and antioxidant enzyme activities] and lipid peroxidation (plasma malondialdehyde and ex vivo oxidation of LDL) were determined. In a subgroup of 10 men, lipoprotein carotenoids were measured. The consumption of carotenoid-rich vegetables significantly increased selected carotenoids in lipoproteins but had only minor effects on their relative distribution pattern. Tomato juice consumption reduced plasma thiobarbituric acid reactive substances (TBARS) by 12% (P: < 0.05) and lipoprotein oxidizability in terms of an increased lag time (18%, P: < 0.05). Carrot juice and spinach powder had no effect on lipid peroxidation. Water-soluble antioxidants, FRAP, glutathione peroxidase and reductase activities did not change during any study period. In evaluating the low carotenoid diet, we conclude that the additional consumption of carotenoid-rich vegetable products enhanced lipoprotein carotenoid concentrations, but only tomato juice reduced LDL oxidation in healthy men.

The DNA 'comet assay' as a rapid screening technique to control irradiated food.

The exposure of food to ionizing radiation is being progressively used in many countries to inactivate food pathogens, to eradicate pests, and to extend shelf-life, thereby contributing to a safer and more plentiful food supply. To ensure free consumer choice, irradiated food will be labelled as such, and to enforce labelling, analytical methods to detect the irradiation treatment in the food product itself are desirable. In particular, there is a need for simple and rapid screening methods for the control of irradiated food. The DNA comet assay offers great potential as a rapid tool to detect whether a wide variety of foodstuffs have been radiation processed. In order to simplify the test, the agarose single-layer set-up has been chosen, using a neutral protocol. Interlaboratory blind trials have been successfully carried out with a number of food products, both of animal and plant origin. This paper presents an overview of the hitherto obtained results and in addition the results of an intercomparison test with seeds, dried fruits and spices are described. In this intercomparison, an identification rate of 95% was achieved. Thus, using this novel technique, an effective screening of radiation-induced DNA fragmentation is obtained. Since other food treatments also may cause DNA fragmentation, samples with fragmented DNA suspected to have been irradiated should be analyzed by other validated methods for irradiated food, if such treatments which damage DNA cannot be excluded.

[International cooperation on the detection of irradiated food]. / Internationale Zusammenarbeit zum Nachweis bestrahlter Lebensmittel.

A survey over recent international developments to detect the irradiation treatment of foods is given, in particular the programmes of "ADMIT" (FAO/IAEA) and of BCR (European Community). The need to detect radiation treatment by analysing the food itself is desirable to check compliance with existing regulations, such as the enforcement of labelling and control of prohibition, to enhance consumer confidence in the correct application of radiation processing, and to protect consumers' freedom of choice between irradiated or unirradiated food products. Some larger collaborative studies on an international scale have already taken place, e.g. ESR measurements of bones from chicken, pork, beef, frog legs and fish, thermoluminescence of insoluble minerals isolated from herbs and spices, gas chromatographic analysis of hydrocarbons and alkylcyclobutanones derived from the lipid fraction of chicken and the microbiological DEFT/APC procedure for spices. These methods could soon be implemented in international standard protocols.

Vitamin E deficiency in rabbits receiving a high PUFA diet with and without a non-absorbable antioxidant. II. Incorporation of 14C-labelled glycine and L-leucine into liver and plasma proteins.

As determined by in vivo studies using [1-14C] L-leucine and [1-14C] glycine, vitamin E deficiency in young rabbits caused a higher turnover rate of liver proteins and of plasma albumin and globulin fractions. This effect was most clearly and consistently observed in animals fed a diet containing 10 mg of the non-absorbable polymeric antioxidant Anoxomer per g of fat in the diet.

Structural investigation of radiation-induced aggregates of ribonuclease.

Following irradiation of bovine pancreatic ribonuclease in aqueous solution with 60Co gamma-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, non-covalent crosslinking also plays a role in the radiolysis of ribonuclease. Thin-layer gel filtration with and without 6 M urea and 2 per cent beta-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40-45 per cent, whereas the remaining 55-60 per cent of the dimers must be linked by other covalent bonds. The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of gamma-irradiated ribonuclease.

[Radiation-induced aggregation of proteins: binding of amino acids to myoglobin (author's transl)]. / Strahleninduzierte Aggregatbildung von Proteinen: Bindung von Aminosäuren an Myoglobin.

When myoglobin is irradiated in the presence of amino acids, the most radiation-reactive species, like the aromatic and sulfur-containing amino acids, will bind preferentially to the protein. The radiation-induced binding is strongly dependent on the concentration of protein and amino acid. Subsequent to irradiation of myoglobin in the presence of radioactively labelled tryptophan followed by tryptic hydrolysis, only a single radioactive spot was detected on the fingerprint. The binding of amino acids is thus not randomly distributed over the protein molecule but occurs at specific reactive sites.

Structural damage of gamma-irradiated ribonuclease revealed by thin-layer isoelectric focusing.

Ribonuclease, irradiated with 60Co gamma-rays in dilute aqueous solution or in the dry state, has been investigated with respect to its charge and size properties. Thin-layer isoelectric focusing revealed extensive change in irradiated RNase; new enzymatically-active components, mainly with isoelectric points lower than in unirradiated RNase were observed. Thin-layer gel chromatography indicated the formation of aggregates which are partially active enzymatically. Aggreation depended on enzyme concentration and was less in more dilute solutions-- at equal degrees of inactivation. Structural damage in the so-called 'native' monomers was revealed by thin-layer isoelectric focusing, their charge properties being distinctly modified.

Fractionation of horseradish peroxidase by preparative isoelectric focusing, gel chromatography and ion-exchange chromatography.

Horseradish peroxidase has been fractionated by preparative isoelectric focusing in a density gradient and in a layer of granulated gel using pH-3-10 and narrow-pH-range carrier ampholytes at different total enzyme loads. The resolution of peroxidase isoenzymes in preparative-layer isoelectric focusing was comparable to that obtained by analytical thin-layer isoelectric focusing. Isoelectrically homogeneous isoenzymes could be isolated with good recovery in a single fractionation step. Despite the excellent separation of the individual isoenzymes by isoelectric focusing in gel layers, an effective purification, indicated by the absorbance ratio A403mn/A278nm, could not be achieved by focusing applied as a single step. By different fractionation sequences combining gel chromatography, ion-exchange chromatography, and isoelectric focusing, individual isoenzymes with a high purity and homogeneous with respect to their size and charge properties have been isolated.