The consequences of physical post-treatments (microwave and electron-beam) on food/packaging interactions: A physicochemical and toxicological approach.

The safety of microwave and electron-beam treatments has been demonstrated, in regards to the formation of reaction products that could endanger human health. An integrated approach was used combining the potential toxicity of all the substances likely to migrate to their chemical characterizations. This approach was applied to polypropylene (PP) films prepared with a selection of additives. Components were identified by liquid and gas chromatography using a mass selective detector system. Their potential toxicity was assessed using three in vitro short-term bioassays and their migrations were carried out using a standards-based approach. After the electron-beam treatment some additives decomposed and there was a significant increase in the polyolefin oligomeric saturated hydrocarbons concentration. PP prepared with Irgafos 168 led to a significantly strong cytotoxic effect and PP prepared with Irganox 1076 induced a dose-dependant estrogenic effect in vitro. Migration values were low and below the detection limit of the analytical method applied.

The Use of Autologous Mesenchymal Stem Cells for Cell Therapy of Patients with Amyotrophic Lateral Sclerosis in Belarus.

We studied a new method of treatment of amyotrophic lateral sclerosis with autologous mesenchymal stem cells. Autologous mesenchymal stem cells were injected intravenously (intact cells) or via lumbar puncture (cells committed to neuronal differentiation). Evaluation of the results of cell therapy after 12-month follow-up revealed slowing down of the disease progression in 10 patients in comparison with the control group consisting of 15 patients. The cell therapy was safe for the patients.

[Highly efficient transfer and stable expression of two genes upon lentivirus transduction of mesenchymal stem cells from human bone marrow].

The efficiency of human bone marrow (BM) mesenchymal stem cell (MSC) transduction with a bicistronic lentivirus vector was estimated, and the stability of transgene expression in genetically modified MSCs was determined. First-passage BM MSCs were capable of efficient transduction with the bicistronic lentivirus vector. The transduction efficiency depended on the multiplicity of infection (MOI), being 64 +/- 6.5 and 88.6 +/- 2.9% at MOI 10 and 20, respectively. The lentivirus transduction efficiency proved independent on the number of passages of a BM MSC culture, and expression of the egfp and dsRed1 transgenes in genetically modified MSCs remained stable for one month of culturing. A comparison showed that the level of egfp and dsRed1 transgene expression was preserved upon hepatogenic differentiation in vitro. The results provide a basis for further development of multigenic modification of human BM MSCs for research and/or therapeutic purposes.

Diazotrophic microbial community of coastal microbial mats of the southern North Sea.

The diazotrophic community in microbial mats growing along the shore of the North Sea barrier island Schiermonnikoog (The Netherlands) was studied using microscopy, lipid biomarkers, stable carbon (δ(13) C(TOC) ) and nitrogen (δ(15) N) isotopes as well as by constructing and analyzing 16S rRNA gene libraries. Depending on their position on the littoral gradient, two types of mats were identified, which showed distinct differences regarding the structure, development and composition of the microbial community. Intertidal microbial mats showed a low species diversity with filamentous non-heterocystous Cyanobacteria providing the main mat structure. In contrast, supratidal microbial mats showed a distinct vertical zonation and a high degree of species diversity. Morphotypes of non-heterocystous Cyanobacteria were recognized as the main structural component in these mats. In addition, unicellular Cyanobacteria were frequently observed, whereas filamentous heterocystous Cyanobacteria occurred only in low numbers. Besides the apparent visual dominance of cyanobacterial morphotpyes, 16S rRNA gene libraries indicated that both microbial mat types also included members of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides group as well as diatoms. Bulk δ(15) N isotopes of the microbial mats ranged from +6.1‰ in the lower intertidal to -1.2‰ in the supratidal zone, indicating a shift from predominantly nitrate utilization to nitrogen fixation along the littoral gradient. This conclusion was supported by the presence of heterocyst glycolipids, representing lipid biomarkers for nitrogen-fixing heterocystous Cyanobacteria, in supratidal but not in intertidal microbial mats. The availability of combined nitrogen species might thus be a key factor in controlling and regulating the distribution of the diazotrophic microbial community of Schiermonnikoog.

Safety evaluation of food contact paper and board using chemical tests and in vitro bioassays: role of known and unknown substances.

In vitro toxicological tests have been proposed as an approach to complement the chemical safety assessment of food contact materials, particularly those with a complex or unknown chemical composition such as paper and board. Among the concerns raised regarding the applicability of in vitro tests are the effects of interference of the extractables on the outcome of the cytotoxicity and genotoxicity tests applied and the role of known compounds present in chemically complex materials, such as paper and board, either as constituents or contaminants. To answer these questions, a series of experiments were performed to assess the role of natural substances (wood extracts, resin acids), some additives (diisopropylnaphthalene, phthalates, acrylamide, fluorescent whitening agents) and contaminants (2,4-diaminotoluene, benzo[a]pyrene) in the toxicological profile of paper and board. These substances were individually tested or used to spike actual paper and board extracts. The toxic concentrations of diisopropylnaphthalenes and phthalates were compared with those actually detected in paper and board extracts showing conspicuous toxicity. According to the results of the spiking experiments, the extracts did not affect the toxicity of tested chemicals nor was there any significant metabolic interference in the cases where two compounds were used in tests involving xenobiotic metabolism by the target cells. While the identified substances apparently have a role in the cytotoxicity of some of the project samples, their presence does not explain the total toxicological profile of the extracts. In conclusion, in vitro toxicological testing can have a role in the safety assessment of chemically complex materials in detecting potentially harmful activities not predictable by chemical analysis alone.

Test procedures for obtaining representative extracts suitable for reliable in vitro toxicity assessment of paper and board intended for food contact.

This paper describes the use of a suite of extraction procedures applicable to the assessment of the in vitro toxicity of paper/board samples intended for food-contact applications. The sample is extracted with ethanol, water, or exposed to modified polyphenylene oxide (Tenax) for fatty, non-fatty and dry food applications, respectively. The water extracts are directly suitable for safety assessment using in vitro bioassays. The ethanol extracts of the paper/board and of the exposed Tenax require pre-concentration to give acceptable sensitivity. This is because the in vitro bioassays can tolerate only a small percentage of added organic solvent before the solvent itself inhibits. The extraction procedures have been selected such that they mimic the foreseeable conditions of use with foods and that they are also fully compatible with the battery of in vitro biological assays for the safety assessment of the total migrate. The application of the extraction protocols is illustrated by the results for one of the many paper/board samples provided by the BIOSAFEPAPER project industrial platform members. The assessment indicated that this sample should not be considered as suitable for use with fatty foodstuffs but was suitable for dry and non-fatty foods. Information subsequently received from the manufacturer revealed that this was a non-food-grade product included in the project to test the capabilities of the bioassay procedures. The selection criteria for the test conditions and the suite of methods developed have been prepared in Comité Européen de Normalisation (CEN) format and is currently being progressed by CEN/TC172 as a European Standard.

In vitro growth of human umbilical blood mesenchymal stem cells and their differentiation into chondrocytes and osteoblasts.

Conditions for culturing and differentiation of human umbilical blood mononuclear cells in vitro were studied. The growth of mesenchymal stem cells was attained in 31 of 54 (57.4%) umbilical blood samples and morphological and immunophenotypical authenticity of these cells was confirmed. Stimulatory effects of 20% AB(IV) human serum and transforming growth factor-beta (TGF-beta) on the growth of mesenchymal stem cells were demonstrated. Osteogenic cells formed in the presence of differentiation factors ascorbic acid, dexamethasone, and beta-glycerophosphate, while chondrogenic cells developed in the presence of dexamethasone, ascorbic acid, and TGF-beta. Differentiation of mesenchymal stem cells was confirmed by histochemical and molecular genetic tests.

The BIOSAFEPAPER project for in vitro toxicity assessments: preparation, detailed chemical characterisation and testing of extracts from paper and board samples.

Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

Safety assessment of food-contact paper and board using a battery of short-term toxicity tests: European union BIOSAFEPAPER project.

An European Union (EU)-funded project QLK1-CT-2001-00930 (BIOSAFEPAPER) involves the development, validation and intercalibration of a short-term battery of toxicological tests for the safety assessment of food-contact paper and board. Dissemination of the results to industry, legislators (e.g. DG Consumer Protection, DG Enterprises, DG Research), standardization bodies such as CEN, and consumers will create an agreed risk evaluation procedure. The project involves pre-normative research in order to establish a set of in-vitro cytotoxicity and genotoxicity tests that will be easily adaptable to food-contact fibre-based materials and have endpoints relevant to consumer safety, including sub-lethal cellular events. These tests will be performed on samples representing actual migration conditions from food-contact paper and board with respect to different foodstuffs, and should form an experimental basis for scientifically sound recommendations for a harmonized system of risk evaluation and product testing.

Regulation of the ban gene containing operon of prophage P1.

A physical map of the ban gene of P1 and sites relevant to its regulation has been deduced from cloning of the appropriate regions of P1 wild-type and of P1 ban regulatory mutants. The cloning required the presence of P1 repressor in the cell confirming the existence of a repressible ban operon (Austin et al. 1978). Evidence for additional member(s) of that operon is presented. Of particular interest for understanding the regulation of ban are the relative positions of a binding site for the P1 repressor and of the regulatory mutations bac and crr that render ban expression constitutive. The results reveal a repressible operon-like structure of about 4 kb within the P1 EcoRI-3 fragment that comprises a c1 repressor binding site/bac - additional gene(s) - crr/ban in the clockwise direction of the circular map of P1.