L. Turco

Caco-2/tc7 cell line characterization for intestinal absorption : how reliable is this in vitro model for the prediction of the oral dose fraction absorbed in human?

Caco-2 cell line is one of the most used in vitro model to study intestinal absorption of compounds at screening level. Several clones have been isolated from Caco-2 cell line and characterized for their activities. Among them, TC7 clone was isolated from a late passage of the parental Caco-2 line and has shown to consist of a more homogeneous population with respect to the most representative functions of the small intestinal enterocytes, with more developed intercellular junctions. On the basis of these characteristics, it was selected within the framework of the EU A-Cute-Tox project to check its suitability to predict intestinal transport. In the present study, drugs, synthetic or natural chemicals have been characterized for their absorption profile in TC7 cells cultivated on semi-permeable filters for 21 days. The absorption experiments have been performed with the highest nontoxic concentration as determined in a preliminary set of cytotoxicity tests. The apparent permeability coefficient (P(app)) has been extrapolated by calculating the passage of the test compound from the donor to the receiver compartment as a time function. The samples have been collected at different time intervals and the concentration of the test compounds analyzed by analytical methods (HPLC, GC, GC/MS). The P(app) obtained with the TC7 clone are comparable to those obtained with the parental cell line. However, some drawbacks related to the experimental system have been highlighted (i.e. low mass balance, adsorption to the plastics), on the basis of which some compounds were excluded from the analysis. In order to check the predictability of the model, a regression analysis has been performed by plotting P(app) values vs. the fraction absorbed in humans (FA, expressed as % of the administered dose). Additional elaborations have highlighted that the specific absorption pathway (passive, active and carrier-mediated) and other factors (i.e. efflux proteins and/or metabolic activity) can strongly affect the robustness of the prediction model. On the basis of the obtained results, TC7 clone has shown to be a model for passive diffusion as reliable as the parental cell line. However, we have remarked the non-suitability of the TC7 cells to predict intestinal absorption: (i) for highly lipophilic compounds; (ii) for poorly absorbed compounds; or (iii) when transporter-mediated routes and/or first pass metabolism are involved. The preliminary study of those factors likely influencing compound biokinetics, as well as the characterization of the cellular model with respect to metabolic and transporter competence, would help in the interpretation of data.

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 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.

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.

The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

On-line EPR study of free radicals induced by peroxidase/H2O2 in human low-density lipoprotein

The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 degrees C and spin trapping techniques to study radical species formed during horseradish peroxidase/H(2)O(2)-initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the alpha-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with beta-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase-mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1-10 microM RSV is able to accelerate alpha-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity.

Caco‐2 Cells as a Model for Intestinal Absorption

The Caco‐2 cell system, a well characterized intestinal in vitro model, makes it possible to evaluate the ability of chemicals to cross the intestinal barrier, as well as to study their transport mechanisms. Permeability values estimated with this model correlate well with human in vivo absorption data for many drugs and chemicals. As a consequence, the use of the Caco‐2 cellular model as a permeability assay to predict oral absorption in humans is growing, and its importance is increasing as a screening tool in drug‐discovery strategies for the prediction of intestinal drug permeability. Strict observance of the experimental conditions, coupled with careful control of the cell‐culture procedures, are crucial for obtaining a meaningful correlation with in vivo data. Because of the intrinsic variability of this absorption model, it is important to highlight that each experimental system must be calibrated with internal reference compounds, to permit data sharing between different laboratories.Curr. Protoc. Toxicol. 47:20.6.1‐20.6.15.

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.

Apoptosis evaluation in epithelial cells exposed to different chemicals: relevance of floating cells

The recent increase in understanding of cell death has promoted new approaches in toxicological studies, mainly those dealing within vitro systems where the evaluation of cell death has been the most widely adopted end-point in measuring the effects of chemical toxicants. The aim of this study was to investigate the possibility of improving the traditional cytotoxicity test protocols in order to produce more specific information on the type of cell death induced by exposure to toxicants. In particular, we characterized the mode of cell death in an established epithelial cell line, HEp-2 cells, which is frequently used in cytotoxicity testing owing to its easy handling and standardization of culture conditions. Reference chemicals for apoptosis and necrosis were selected as controls, together with other molecules that have been shown, in preliminary studies, to induce various morphological and structural modifications in relation to cell death. The results obtained show that: (a) the floating fraction of treated cells gives the clearest picture of the necrotic/apoptotic distribution; (b) morphological analysis is crucial for characterization of apoptosis; (c) more than one cytotoxic end-point is necessary to clearly identify the type of cell death.