bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Liver Slices Express the Same Lidocaine Biotransformation Rate as Isolated Human Hepatocytes
Authors: Olinga Peter , Merema Marjolijn T. , Meijer Dick K.F. , Slooff Maarten J.H. , Groothuis Geny M.M.
Published: August 2020

In order to investigate whether liver slices are a valuable tool for the assessment of drug metabolism in human liver, we compared the phase I metabolism of lidocaine in human liver slices and hepatocytes prepared from three human livers. Lidocaine is mainly metabolised to monoethylglycinexykdide (MEGX) via a cytochrome P450-mediated N-deethylation. The results indicate that the three livers showed considerable inter-individual differences in the rate of formation of MEGX, and that this difference was equally reflected in slices and isolated cells. The use of liver slices is still under development, and optimal incubation conditions still need to be assessed. However, these results suggest that, in slices of 200–300μm thickness, virtually all hepatocytes are involved in the biotransformation of lidocaine, and that the metabolic activity is preserved equally well as in isolated hepatocytes.
Similarities in the Mechanisms of Antibacterial Activity (Microtox™ Assay) and Toxicity to Vertebrates
Authors: Rosenkranz Herbert S. , Pangrekar Jyotsna , Klopman Gilles
Published: August 2020

The structural determinants associated with inhibition of bacterial growth (Microtox assay), as well as those associated with other toxicological activities, were identified using the CASE/Multi-CASE structure-activity relational system. CASE/Multi-CASE also possesses the ability to compare structural determinants associated with different biological phenomena. The extent of the homology between structural determinants can be used to evaluate common mechanisms of action. Using this approach, significant commonalities between structural determinants associated with the Microtox assay and those associated with toxicity to mammals and fish were found, suggesting that there may be a mechanistic relationship between these toxicological activities. The findings indicate that, within these structural similarities, the Microtox assay can be used as an indicator of potential toxicity to mammals and fish.
Preliminary Findings of an In Vitro Assay Based on Human Complement Activation for the Detection of Chemicals with Phototoxicity Potential
Authors: Sladowski Dariusz , Steer Sarah , Moore Lynda , Glassborow Ruth , Clothier Richard , Balls Michael
Published: August 2020

Eight known photosensitising chemicals and one non-photoaensitiser were compared for their ability to activate the complement system of human serum in both the presence and the absence of ultraviolet (UV) radiation. Commercially available ELISA systems for the detection of C4d and iC3b fragments were used to investigate the classical and alternative pathways, respectively. The results indicated that, whereas the classical pathway was unaffected, all the photosensitisers tested were capable of activating the alternative pathway in the presence of UV light, and that this activation occurred at different levels, depending upon the chemical itself. The non-photosensitising chemical, sodium dodecyl sulphate, acted as a negative control. It is, therefore, suggested that this type of method could be considered for inclusion in any battery of in vitro tests designed for the detection of potential photosensitising chemicals.
The Galileo Data Bank on Toxicity Testing with In Vitro Alternative Methods. II. Toxicology Profiles of 20 Chemicals
Authors: Loprieno Nicola , Boncristiani Guido , Bosco Elena , Nieri Maria , Loprieno Gregorio
Published: August 2020

The identification of the hazard of chemicals to man has relied on the use of several animal models. However, the availability of various cell toxicity models as alternatives to the use of animals has stimulated attempts to evaluate in vitro data for use in the prediction of human toxicity.

The cell toxicity models developed previously are capable of indicating a variety of endpoints susceptible to the activity of various chemical substances.

The in vitro data derived so far from testing a variety of types of chemicals, have been used to develop toxicology profiles for twenty chemicals, which are presented in this paper. Data have been selected from among those already entered in the Galileo Data Bank, a computerised data system containing all the available existing data derived using in vitro methods.
Cell Agglutinating Activity of A-Gliadin-Related Synthetic Peptides
Authors: Vincenzi Massimo De , Dessi Maria Rita , Giovannini Claudio , Cantafora Alfredo , Pavone Vincenzo
Published: August 2020

Previous studies have suggested that various A-gliadin-derived peptides actively agglutinate K562CS) cells. These active peptides showed the following common sequences: pro-ser-gln-gln and gln-gln-gln-pro. In this study, we have synthesised and tested the following toxic fragments: the peptide with the 31–55 amino acid sequence, which contains both the toxic sequences, and the peptides 31–43 and 44–55, which contain the sequences gln-gln-gln-pro, and pro-ser-gln-gln, respectively. Both the peptides with either the gln-gln-gln-pro or pro-ser-gln-gln sequences were active in agglutinating all cells. However, the peptide 44–55 agglutinated 100% of the cells at a concentration two times greater than the peptide 31–43. This suggests a relationship between the gln-gln-gln-pro and pro-ser-gln-gln sequences and the damaging effect of gliadins on the coeliac small intestine in individuals affected by coeliac disease. Moreover mannan and oligomers of N-acetylglucosamine were found to be able to prevent the cell-agglutinating activity of the active peptides.
Toxicity of 39 MEIC Chemicals to Bioluminescent Photobacteria (The Biotox™ Test): Correlation with Other Test Systems
Authors: Kahru Anne , Borchardt Barbara
Published: August 2020

Photobacterium phosphoreum are naturally luminescent marine bacteria, which emit light as a result of metabolism. Any damage to the cellular metabolism caused by a toxic substance can therefore be monitored by measuring the change in light output of the bacteria, the light loss being proportional to the degree of toxicity. The Biotox™ test, based on reconstituted freeze-dried (viable) Photobacterium phosphoreum as the test organism, was used for the short-term toxicity testing of 39 chemicals from the MEIC list. Concentration-effect curves for these 39 chemicals were measured and five-minute EC50 values (the concentration of chemical, mM, which reduces the light output of the bacteria by 50% after contact for five minutes) were calculated. The EC50 data from the Biotox test were correlated with data from the literature: octanol/water partition coefficients, EC50 data from the Microtox™ test, minimal inhibitory concentration values for Escherichia coli and Bacillus subtilis and acute toxicity data from animal and human cell lines, rodents and man. The correlation coefficients ranged between 0.63 and 0.93.
In Vitro Transformation of C3H/10T1/2 and SHE Cells by Propylene Oxide and Epichlorohydrin
Authors: Kolman Ada , Dušinská Mária , Slameňová Darina
Published: August 2020

Neoplastic cell transformation by the carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH), was investigated in two mammalian assays involving murine C3H/10T1/2 cells and Syrian hamster embryo (SHE) cells. In C3H/10T1/2 cells, PO increased the frequency of morphological transformations 9–18 times compared to the background frequency, at concentrations of 2.5–10mM (1-hour treatment). In SHE cells, a similar increase (5–15 times) was reached at concentrations of 5-20mM (8-day treatment). In both assays, ECH increased the frequency of morphological transformations only at the highest exposure levels (2mM for C3H/10T1/2 cells and 0.5mM for SHE cells).
Aquatic Toxicology in Vitro: A Brief Review
Authors: Isomaa Boris , Lilius Henrik , Råbergh Christina
Published: August 2020

There is an urgent need for effective in vitro tests in aquatic toxicology, because only a very small proportion of the chemicals in common use have been adequately tested for their toxicity to aquatic organisms and aquatic ecosystems. Toxicity tests with higher animals, besides being time-consuming and expensive, are ethically questionable, which further increases the importance of developing efficient in vitro toxicity tests. In developing in vitro tests for toxicity assessments, aquatic toxicology lags behind mammalian toxicology. Aqueous environmental chemistry is complex, and the sensitivity of the organisms living in a particular aquatic environment may vary considerably. The predictive value of single-species or cell culture tests is therefore generally considered to be low. Nevertheless, single-species tests, utilising bacteria, algae, protozoans and invertebrates, have frequently been used in in vitro toxicity studies of aquatic pollutants (mainly as screening tests). Attempts at large-scale validations are few. Such attempts seem to be hampered by the complexity of the aquatic ecosystem. Although cells from aquatic organisms have been isolated and cultured for many years, the use of isolated or cultured cells in aquatic toxicology has been limited. However, during the last few years, interest in the use of fish cells in toxicity testing has grown rapidly. For aquatic in vitro toxicology to develop further, a more comparative and mechanistic approach needs to be adopted.
NMR Imaging of Laboratory Mice at 2 Tesla: A Non-invasive Insight into Normal Anatomy
Authors: Munasinghe Jeeva P. , Carpenter T. Adrian , Hall Laurance D.
Published: August 2020

Many anatomical features of the mouse which are of interest in developmental biology, lie in the sub-millimetre range and can be visualised by NMR imaging at 2 Tesla (T) with spatial resolution in the range of 180–250μm for a slice thickness of 1–1.5mm. Using surface coils to localise a region of interest close to the surface, a resolution as high as 100μm can be achieved. It is possible to obtain a series of up to 20 slice images with a total imaging time of less than 30 minutes.
Species-specific Effects of Nitromethylene Heterocycle Insecticides on Nicotinic Receptors in Locust Neurons and Mouse N1E-115 and BC3H1 Cells
Authors: Oortgiesen Marga , Zwart Ruud , Vijverberg Henk P.M.
Published: August 2020

The effects of nitromethylene heterocycle (NMH) insecticides on subtypes of nicotinic acetylcholine (nACh) receptors were investigated in locust thoracic ganglion neurons, mouse N1E-115 neuroblastoma cells, and mouse BC3H1 muscle cells by using electrophysiological techniques. In locust neurons, all of the six NMH insecticides tested induced transient inward currents resembling nicotinic ACh-induced inward currents, while, in the continued presence of the NMH compounds, the ACh-induced inward current was blocked. The amplitude of the inward current and the blocking effects of the NMH insecticides were enhanced by concentrations between 0.1 and 10μM. Cross-desensitisation with the ACh-induced inward current confirmed that the NMH-induced inward current was governed by the activation of nACh receptors. Mammalian endplate type nACh receptors in BC3H1 cells and mammalian neuronal type nACh receptors in N1E-115 cells were much less sensitive to the NMH insecticides than the locust neuronal nACh receptors. At a concentration of 10μM, which blocked 80–100% of the ACh-induced inward current in locust neurons, NMH insecticides only partially blocked the ACh-induced inward currents mediated by the two subtypes of mammalian nACh receptors. NMH insecticides also failed to induce significant agonist effects in the mammalian cells at this concentration.

The results provide a possible explanation for the selectively greater toxicity of NMH insecticides to insects than to vertebrates, at the level of nACh receptor subtypes and, hence, demonstrate that this in vitro approach is valuable for the investigation of species-specific interactions of compounds at their target site.
The Fund for the Replacement of Animals in Medical Experiments (FRAME): The First 25 Years
Authors: Annett Bill
Published: August 2020

The establishment of FRAME in 1969 and the Charity's development during its first 25 years are reviewed, with emphasis on FRAME'S campaigns, FRAME publications, the FRAME Research Programme, the FRAME Toxicity Committee, and FRAME'S role in the drafting and passage through Parliament of the Animals (Scientific Procedures) Act 1986.
The Public Debate about Animal Experimentation
Authors: Matfield Mark
Published: August 2020

The nature of campaigning against animal experimentation and its effects on public opinion and laboratory animal welfare are examined. A three-part model is proposed in which the activities of campaigning groups form the motivation phase which, in some circumstances, can produce the second phase: change of public attitudes. This leads to the third phase: making the change, in which the animal researchers, technicians and veterinarians change their practice. The role of organisations promoting the Three Rs is incorporated by viewing their role as catalysing the progression through the three phases. Some of the consequences of this model for the development of in vitro alternatives are examined.
A New Reductionism for Animal Research
Authors: Fisher Chris
Published: August 2020

This article examines the decline in animal experimentation observed in the last 25 years. It considers possible reasons for this and its significance. However, it also asks whether more could have been achieved, and considers what needs to be done to ensure that the current rate of decline is continued and increased — not just in the UK, but also in Europe and beyond. It is concluded that future reductions in animal experimentation need to be a matter of policy, rather than the product of circumstance. Furthermore, it is concluded that policy objectives alone will be insufficient unless supported by a clear strategy and adequate resources for effective implementation.
The Third R: Refinement
Authors: Rowan Andrew N.
Published: August 2020

This review attempts to provide an introduction to the complicated subject of refinement, the third R in the concept of alternatives. It starts with a brief discussion of what refinement means and the lack of specific attention paid to this third R. This is followed by an analysis of the conceptual underpinnings of pain, distress and suffering, and the problems of both definition and measurement which must be faced if we are to be objective and consistent in our search for refinement. The review then touches upon husbandry, care and handling issues as they affect animal discomfort and distress. Antibody production, both polyclonal and monoclonal, is discussed as an example of the refinement of research techniques. Finally, a few brief comments are offered on the refinement of a variety of other experimental techniques, including those used in toxicology, cancer research and behavioural research.
Supply, Coding and Distribution of Samples for Validation Studies
Authors: Brantom Paul G. , Aspin Pat , Thompson Carol
Published: August 2020

The procedures followed for coding, labelling and distribution of samples for blind validation trials are described, paying particular attention to those elements of the process which preserve the blind nature of the trial, while ensuring that the sample identity is that intended.
Transgenesis and Animal Welfare
Authors: Moore Colin J. , Mepham T. Ben
Published: August 2020

The two main techniques used in biomedical research for the production of transgenic animals have several implications for animal welfare in terms of the Three Rs of Russell & Burch. Some are intrinsic to the transgenic objectives, while others relate to the effects of mutations, transgene expression, associated methodologies, and husbandry or production systems. All of these actual and potential implications for animal welfare demand serious consideration within a broad ethical analysis of the technology. In the light, of the Three Rs, this may require a fundamental reappraisal of the processes by which such scientific procedures are approved.
Chimpanzee Biomedical Experiments: A Question of Efficacy
Authors: Fouts Roger
Published: August 2020

The efficacy of biomedical research on chimpanzees is addressed from an empirical point of view. The confounding effects of “standard housing” on experiments are examined, with regard to deleterious effects on the neurophysiological makeup of the subjects and thus the effects on the results of any research examining neurological or immunological factors. Also, specious symptom similarity can cause one to assume a common aetiology, which is often unwarranted. The assumption of common reactions from different species violates sound scientific procedures. The high probability of Type 1 errors in the chimpanzee AIDS model is discussed. Also, unjustified generalisation across species is discussed. Finally, the rational and ethical problems surrounding the use of chimpanzees in biomedical research are discussed.
Why is it Unethical to use Chimpanzees in the Laboratory?
Authors: Goodall Jane
Published: August 2020

Chimpanzees are more like humans than any other living beings, differing in the composition of their DNA by just over one per cent. There are striking similarities in the anatomy and wiring of the chimpanzee and human brains and central nervous systems.

Thus, it should not be surprising to find that there are also striking similarities in the social behaviour, emotional needs and expressions, and cognitive abilities of chimpanzees and humans. These similarities have become increasingly apparent during the last 15 years. Chimpanzees in the wild develop close affectionate bonds between family members that may persist throughout their lifetime of 50 years or more, and examples of true altruism, when individuals protect or even save the lives of non-related companions. Chimpanzees use many objects as tools, and tool-using behaviours differ from place to place across their range. Indeed, there are a number of behaviours that vary between different groups — evidence of cultural traditions passed from one generation to the next through observational learning and imitation.

Thus, chimpanzees have a very special relationship with humans. A healthy adult chimpanzee is more similar to a healthy adult human in the expression of the intellect than a brain-damaged human, yet in many medical research facilities, chimpanzees are maintained in bleak, bare cages measuring only 5’ x 5’ x 7'. They may remain in these prisons for life. We do not treat hardened human killers so badly in our society today—there would be a public outcry if we did.

I feel strongly that the use of a being so like us, as a human guinea-pig, is not morally justified, and to that end the Jane Goodall Institute has been involved in three workshops with the aim of clarifying the extent to which they are seen to be useful in diseases such as hepatitis and AIDS research. There is no consensus among scientists regarding their usefulness at the present time. If the proposed experiments of transplanting chimpanzee bone marrow tissue into AIDS patients go ahead in the Netherlands, it will be a sad blow for chimpanzee liberation.

The attitude of those who believe that any use of non-human primates can be justified provided it results in some benefit, or expected benefit, to humankind, is of precisely the same mind set as that which once allowed us to exploit human beings of another race and use them as slaves. Once we admit that chimpanzees have minds and feelings, are capable of sadness, fear and despair, are able to feel pain, show altruism, and are capable of communicating with each other and with humans in a man-made language, we have to ask serious questions, initially of ourselves, as to whether we should continue to use them in medical research.
Moral Issues in Relation to Chimpanzee Field Studies and Experiments
Authors: Reynolds Vernon
Published: August 2020

The ancient Greek roots of two traditions in science are traced. The first, derived from Pythagoras, emphasises continuity of life-forms and respect for animals. The second, derived from Aristotle, divides humans from animals because humans possess reason whereas animals lack it. This gives humans the right to use animals for their own purposes. Primate field workers are closer to the former tradition than the latter, but rarely involve themselves in detailed consideration of animal experiments. With the post-Darwinian awareness of the continuity of all life-forms, it is now known that chimpanzees, in particular, are very close to humans in most respects, and it is argued that their use in invasive experiments is no longer morally defensible.
The Evaluation of Pesticide Ingredients and Formulations In Vitro and Correlations with In Vivo Data
Authors: Clothier Richard H. , Morris Joanne , Lankford William T.
Published: August 2020

Pesticides are often insoluble directly in aqueous solvents, but can be dissolved/suspended in surfactant-uased formulations. Both surfactants and pesticides can induce irritation. Since a single in vitro assay has proved inadequate for evaluating the toxicity of a chemical and its ability to cause an irritant response, a combination of assays was employed to examine the potential toxicities of two pesticide formulations. The surfactant-based vehicles had toxicities that reflected their surfactant concentration. The formulation containing 5% permethrin required a more concentrated vehicle than was needed to dissolve 0.1% cypermethrin. In vitro, the ID50 dose (i.e. the dose which inhibited the increase in total cellular protein by 50%) was 576μg/ml for the permethrin formulation and 1080μg/ml for the cypermethrin formulation. This corresponded closely to the ID50 values for the vehicles alone (464μg/ml and 1230μg/ml, respectively). When tested at high concentrations on confluent cells over a 1-minute exposure period to mimic potential exposure of the eye, the more concentrated vehicle, Lanosol 50 ME, was 4–6 times more toxic than Siege II. Technical grade permethrin and cypermethrin had low toxicities in each of the in vitro tests employed. Taken together, these results reflected the in vivo profiles available.
Alternatives in Canada
Authors: Griffin Gilly
Published: August 2020

In Canada, promotion of the Three Rs concept of alternatives occurs largely through the Canadian Council on Animal Care. Institution-based animal care committees are required to conduct an ethical review of all protocols which use animals in research, teaching or testing. Investigators are expected to use animals only when their best efforts to find an alternative have failed. Those using animals are required to employ the most humane methods on the smallest number of animals possible.

The Joseph F. Morgan Research Foundation was established to promote the development and use of alternative methods in Canada. The focus of the Foundation has been on the use of alternative methods for testing purposes. However, the Foundation also encourages the acceptance of each of the Three Rs throughout Canadian science. The Foundation is therefore in the process of developing a refinement alternatives programme.
MEIC Evaluation of Acute Systemic Toxicity
Authors: Clemedson Cecilia , McFarlane-Abdulla Elisabeth , Andersson Marianne , Barile Frank A. , Calleja Mabel C. , Chesné Christophe , Clothier Richard , Cottin Martine , Curren Rodger , Dierickx Paul , Ferro Margherita , Fiskesjö Geirid , Garza-Ocañas Lourdes , Gómez-Lechón Maria Jose , Gülden Michael , Isomaa Boris , Janus Jeffrey , Judge Paula , Kahru Anne , Kemp Richard B. , Kerszman Gustaw , Kristen Udo , Kunimoto Manabu , Kärenlampi Sirpa , Lavrijsen Karel , Lewan Lillemor , Lilius Henrik , Malmsten Anders , Ohno Tadao , Persoone Guido , Pettersson Roland , Roguet Roland , Romert Lennart , Sandberg Maria , Sawyer Thomas W. , Seibert Hasso , Shrivastava Ravi , Sjöström Michael , Stammati Annalaura , Tanaka Noriho , Torres-Alanis Oscar , Voss Jens-Uwe , Wakuri Shinobu , Walum Erik , Wang Xinhai , Zucco Flavia , Ekwall Björn
Published: August 2020

Results from tests of the first 30 MEIC reference chemicals in 68 different toxicity assays are presented as a prerequisite to subsequent in vitro/in vivo comparisons of acute toxicity data. A comparative cytotoxicity study was also carried out. Firstly, the variability of all of the results was analysed by using principal components analysis (PCA), analyses of variance (ANOVAs) and pairwise comparisons of means according to Tukey's method. The first PCA component described 80% of the variance of all of the cytotoxicity data. Tukey's ANOVA indicated a similar sensitivity for the assays, of approximately 80%. Secondly, the influence of five major methodological components on the general variability of the results was evaluated by linear regression and ANOVA linear contrast analyses. The findings were that: a) the toxicity of many chemicals increased with exposure time; b) in general, human cytotoxicity was predicted well by animal cytotoxicity tests; c) this prediction was poor for two chemicals; d) the prediction of human cytotoxicity by the ecotoxicological tests was only fairly good; e) one organotypic endpoint used, i.e. contractility of muscle cells, gave different results to those obtained according to viability/growth toxicity criteria; f) twelve comparisons of similar test systems involving different cell types (including highly differentiated cells) showed similar toxicities regardless of cell type; and g) nine out often comparisons of test systems with identical cell types and exposure times revealed similar toxicities, regardless of the viability or growth endpoint measurement used. Factors b, f and g must be the main causes of the remarkable similarity between the total results, while factors a, c, d and e, together with other minor factors that were not analysed, contributed to the 20% dissimilarity. The findings strongly support the basal cytotoxicity concept, and will facilitate future in vitro toxicity testing.
The Development of Corneal Opacity, Increased Hydration and Increased Thickness in Isolated Porcine Corneas in Response to Timolol Maleate Solutions Containing Various Preservatives
Authors: Igarashi Haruyoshi , Sato Chiharu , Chiba Yu , Ogawa Katsuhiro , Kojima Michio
Published: August 2020

The effects of 0.125%, 0.25% and 0.5% timolol maleate on the opacity, thickness and hydration of isolated porcine corneas were determined. The effects of adding 0.005% benzalkonium chloride, 0.5% chlorbutanol or a combination of 0.026% methyl p-hydroxybenzoate, 0.014% propyl p-hydroxybenzoate and 0.2% chlorbutanol to the timolol solutions were also assessed. Over a 4-hour incubation period, a concentration-related opacity developed in the presence of timolol alone, and was not greatly affected by the addition of the p-hydroxybenzoate mixture. However, the addition of benzalkonium chloride or the higher concentration of chlorbutanol markedly increased opacity. Corneal thickness increased significantly in the presence of 0.25% and 0.5% timolol and even further in the presence of the preservatives, chlorbutanol being the most potent. Hydration increased slightly with time and also in the presence of each of the preservatives. The need to achieve a balance between possible corneal toxicity and effective batericidal activity is discussed.
Toxicity of Bread Wheat Lines Lacking Prolamins Encoded by the Gli-B1/Gli-B5/Glu-B3 and Gli-D1/Glu-D3 Loci in Coeliac Disease as Determined by their Agglutinating Activity
Authors: De Vincenzi Massimo , Dessì Maria R. , Luchetti Roberto , Pogna Norberto , Redaelli Rita , Galterio Giovanni
Published: August 2020

Peptic-tryptic (PT) digests of alcohol-soluble proteins from the flour of three mutant lines of bread wheat, lacking γ-gliadins, γ-gliadins and low molecular-weight glutenin subunits encoded by the Gli-B1/Gli-B5/Glu-B3 loci (line S. Pastore 4A), the Gli-D1/Glu-D3 loci (line Alpe 1I-) or both groups of loci (line DM 22166), were compared with those of the normal cultivars S. Pastore and Alpe 1 I for their agglutinating activities on human myelogenous leukemia K562(S) cells, agglutination being strongly associated with toxicity for the coeliac intestine. All of the genotypes tested contained A-type α-gliadins, which constituted about 19% of the gliadins in the S. Pastore and Alpe 1I cultivars, 24.5% in the S. Pastore 4A and Alpe 1I null lines, and 34.8% in the double mutant line, DM 22166, as determined by densitometric scanning of their acid polyacrylamide gel electrophoresis patterns. The minimal concentrations of PT digest required to agglutinate 100% of K562(S) cells were 73mg/l and 96mg/l, in the S. Pastore and Alpe 1I cultivars, respectively, compared with 146mg/l, 138mg/l and 200mg/l in the “null” lines, S. Pastore 4A, Alpe 1 I- and DM 22166, respectively. The results indicated that proteins other than α-gliadins are involved in the gluten-sensitive enteropathy.
Determination of Phosphofructokinase and Enolase Activities in Cultured Mouse Neuroblastoma Cells: Application to the In Vitro Detection of Neurotoxic Effects
Authors: Andres Maria Isabel , Repetto Guillermo , Sanz Pilar , Repetto Manuel
Published: August 2020

Simple methods for the determination of phosphofructokinase (PFK) and total enolase (END activities in cultured mouse neuroblastoma cells have been developed. The influences of changes in glucose metabolism, induced by chlorpromazine, cycloheximide, 2,4-dinitrophenol and iodoacetic acid, on PFK and ENL activities in vitro were compared as a practical application of the methods. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed for 24 hours and cytotoxic effects were evaluated. All the determinations were carried out in the same 96-well tissue culture plates in which exposure took place. Chlorpromazine and cycloheximide produced opposite effects on both PFK and ENL activities. While chlorpromazine increased PFK activity by 50% and decreased ENL activity by 30%, cycloheximide inhibited PFK activity by 50% and increased ENL activity by 45%. The response to dinitrophenol was quite different; both PFK and ENL activities were increased, by 45% and 35% respectively. After exposure of the cells to iodoacetic acid, neither PFK nor ENL activities showed statistically significant differences from the levels in control cells. The different responses elicited by the four toxicants suggest that the two enzymes selected are useful for differentiating among diverse types of mechanism of action.
The Three Rs in Research and Education: A Long Road Ahead in the United States
Authors: Orlans F. Barbara
Published: August 2020

Attitudes toward the Three Rs concept of refinement, reduction and replacement in the United States in research and education are widely divergent. Positive responses have come from several sources, notably from four centres established to disseminate information about alternatives. Funding sources to support work in the Three Rs have proliferated. The activities of institutional oversight committees have resulted in the nationwide implementation of important refinements. In the field of education, student projects involving pain or death for sentient animals have declined, and the right of students to object to participation in animal experiments on ethical grounds has been widely established. However, there is still a long way to go.

Resistance to alternatives is deep-seated within several of the scientific disciplines most closely associated with animal research. The response of the National Institutes of Health to potentially important Congressional directives on the Three Rs has been unsatisfactory. The prestigious National Association of Biology Teachers, which at first endorsed the use of alternatives in education, later rescinded this policy, because of opposition to it. An impediment to progress is the extreme polarisation of viewpoints between the biomedical community and the animal protectionists.
Relevance of Animal Studies in Regulatory Toxicology: Current Approaches and Future Opportunities
Authors: van de Sandt Johannes J.M. , Feron Victor J.
Published: August 2020

With rapidly increasing knowledge of toxicological processes, the scientific value and relevance of toxicity studies for risk assessment must be re-evaluated. In this paper, it is proposed that the rigid risk evaluation currently required should be replaced by a more flexible, case-by-case approach, in order to increase the relevance of each animal test conducted. The development of new types of toxicity studies and their application in risk evaluation are also described.
Dynamic Qualities of Validation and the Evolution of New In Vitro Toxicological Tests
Authors: Walum Erik , Forsby Anna , Clemedson Cecilia , Ekwall Björn
Published: August 2020

This review summarises some aspects of the dynamics of the evolution of toxicological test methods based on cell biology. Within the multicentre evaluation of in vitro cytotoxicity (MEIC) programme, some general principles for validation have been proposed: a) a human database should be used as the source of reference data in the validation of new methods aimed at predicting human toxicity; b) the relevance of a test should be determined before its reliability is assessed; c) a parallel validation of methods is preferable to a serial validation; and d) toxicokinetic data should be included in the validation process to improve the predictivity of cytotoxicity test results. These toxicokinetic data can be used to extrapolate the cytotoxic in vitro concentrations to provide human toxic exposure levels. As part of test development, the cytotoxic concentration can also be compared directly with the critical toxic human blood or tissue concentration. This approach is being explored in the ERGATT/CFN integrated toxicity testing scheme (ECITTS) prevalidation project. The critical toxic concentrations are determined by using a set of neurospecific cellular tests, chosen and combined on the basis of knowledge of common neurotoxicity mechanisms. Another approach to selecting tests for prevalidation is through the development of tests that are found to be necessary and complementary to existing tests. Such a programme has been initiated on the basis of the results of the MEIC study. The progress made so far in this “missing tests programme” is presented in this paper.
Cholinergic and Glutaminergic Excitation of Neuronal Cells
Authors: Savolainen Kai M. , Tervo Pirkko , Loikkanen Jarkko , Naarala Jonne
Published: August 2020

Excessive cholinergic or glutaminergic brain stimulation may result in seizures, excitotoxicity and neuronal damage. Cholinergic neuronal excitation is mediated via muscarinic receptors which couple with GTP-binding proteins (G–proteins), activate phospholipase C, and produce the inositol lipid second messengers, inositol-1,4,5,-trisphosphate (InsP3) and diacyl-glycerol (DG). InsP3 facilitates intracellular Ca2+ metabolism and DG activates protein kinase C (PKC). Glutaminergic neuronal stimulation is mediated through ionotropic N-methyl-D-aspartate (NMDA) receptors, which increase Ca2+ influx, and kainate α-amino-3-hydroxy-5-methyl-4-isoxalolproprionic acid receptors, which mainly regulate Na+ fluxes. Glutaminergic metabotropic receptors are also coupled to a G-protein, and their stimulation activates neurons through increased production of InsP3 and DG. A salient feature in glutamate-induced excitotoxicity is the induction of an oxidative burst, subsequent oxidative stress, and damage to the neurons. The glutamate-induced oxidative burst can be amplified by lead, a direct activator of PKC, and the oxidative burst can be blocked by a PKC inhibitor, suggesting an important role for PKC. Carbachol also induces an oxidative burst in neuronal cells and this is associated with elevations of free intracellular calcium. The ability of an NMDA receptor antagonist, AP-5, to block carbachol-induced elevations of free intracellular calcium, suggests that activation of muscarinic receptors is associated with a simultaneous glutamate receptor activation. Thus, cross-talk between cholinergic muscarinic and glutaminergic receptors may be an important contributing factor in cholinergic and glutaminergic excitotoxicity.
The Use of Isolated Hepatocytes from Rats with Enzyme-altered Foci: Studies of p53 Expression
Authors: Stenius Ulla , Martens Ulf , Högberg Johan
Published: August 2020

This paper describes a new version of the widely used technique of isolated rat hepatocyte primary culture. Hepatocytes are isolated from carcinogen-treated rats, and the cultures contain two populations of cells. Data indicate that one cell type derives from enzyme-altered foci (EAF) and one from unaltered tissue. EAF are regarded as precarcinogenic lesions, and the model might be useful in establishing mechanistic and test data for the risk assessment of carcinogens. The two populations differ in their responses to many xenobiotics and toxins. They also differ in their responses to endogenous cell growth control factors. Data are presented on p53 expression in cultured hepatocytes in response to exposure to methyl iodide or diethyl nitrosamine. The responses of the two cell types differed. For example, 0.2mM methyl iodide induced p53 expression in 2–4% of the non-EAF cells, but in less than 1% of the EAF cells. 2% of non–EAF cells and 8% of EAF cells incorporated thymidine. It is concluded that many EAF cells have an attenuated expression of p53 in response to DNA-damaging agents. EAF hepatocytes with damaged DNA may replicate DNA, and this effect could be critical for tumour development.
The Influence of Glutathione on the Cytotoxicity of Metals in Rat Hepatoma-derived Fa32 Cells
Authors: Dierickx Paul J.
Published: August 2020

The cytotoxicities of mercury, cadmium, nickel, cobalt, zinc and copper were investigated in rat hepatoma-derived Fa32 cells by using the neutral red uptake inhibition assay with three treatment regimens (2 hours, 24 hours and 1 week). Nickel and cobalt were almost non-toxic after 2 hours. Good correlations were observed between the 24-hour and the 1-week cytotoxicities, and cytotoxicity in human hepatoma-derived Hep G2 cells. L-buthionine-S,R-sulphoximine reduced the glutathione content to 5% after 24 hours. The cytotoxicity of the metals increased (3–12 times) in glutathione-depleted cells. A good agreement was demonstrated by HPLC between the glutathione S-transferase (GST) subunit composition in Fa32 cells and in rat liver, except that subunit 7 is also a major subunit in the hepatoma cell line. No evidence was obtained for an interaction of the GSTs in the glutathione-modulated cytotoxicity of the investigated metals.
Detection of Oxidised Purines and UV-induced Photoproducts in DNA of Single Cells, by Inclusion of Lesion-specific Enzymes in the Comet Assay
Authors: Dušinská Mária , Collins Andrew
Published: August 2020

The comet assay (single cell gel electrophoresis) is a rapid, very sensitive method for the detection of DNA strand breaks at the level of single cells, which is now being applied in genotojricity testing. We modified this method for the detection of a variety of kinds of DNA lesion, by treating nucleoid DNA in the gel with either formamidopyrimidine-DNA glycosylase (which recognises ring opened purines, 8-hydroxyguanine and apurinic/apyrimidinic sites), or uvrABC excinuclease (uvrABC; which has a rather broad specificity, including bulky lesions and UV photoproducts). By using this modified assay, we demonstrate the removal of DNA strand breaks and oxidised purines upon incubating cells after treatment with hydrogen peroxide. This modification clearly increases the usefulness of the assay for the analysis of DNA damage and repair, for screening human populations for DNA damage, and for testing novel chemicals for genotoxicity.
Recommendations for the Application of Biostatistical Methods during the Development and Validation of Alternative Toxicological Methods
Authors: Holzhütter Hermann-Georg , Archer Graeme , Dami Nadia , Lovell David P. , Saltelli Andrea , Sjöström Michael
Published: August 2020

Assessment of the relevance and reliability of alternative toxicological methods requires the application of biostatistical methods. This report contains the recommendations of the ECVAM Task Force on Biostatistics for the appropriate use of biometrical methods, and for closer collaboration between biostatisticians and experimental toxicologists in the development and validation of alternative methods. The main objectives of these recommendations are to improve the compatibility of the results obtained from different studies, and to prevent the misinterpretation of statistical analyses. The following points are discussed: a) the general importance of biostatistics in the development and validation of alternative tests; b) optimisation of study designs; c) statistical characterisation and processing of toxicological data; and d) the analysis of relationships which may exist between data obtained from various tests. In particular, the establishment of appropriate prediction models which are able to predict the toxic effects likely to occur in vivo from the results obtained in an in vitro test system is discussed. The members of the Task Force hope that this report will help to stimulate discussions among biostatisticians and experimental toxicologists about how the proper application of biostatistical methods can improve the design and evaluation of alternative toxicological methods.
The Capacity of Murine Epithelial Cell Lines with Different Degrees of Malignant Transformation to Metabolise Benzo[a]pyrene
Authors: Viezzer Cristina , Revoltella Roberto , Celotti Lucia
Published: August 2020

Cultures of three murine cell lines derived from fetal mouse liver (FL, FLT3 and FLT5) were treated with benzo[a]pyrene to evaluate their ability to activate promutagens. DNA adducts were detected in all three cell lines when they were treated with the major active metabolite of benzo[a]pyrene, benzo[a]pyrene diolepoxide. DNA adducts were also produced in FL and FLT3 cells when they were treated with the promutagen, benzo[a]pyrene, while treatment of FLT5 cells resulted in very few DNA adducts. In treated FL and FLT3 cells, the most evident adduct spot detected showed a chromatographic position similar to that of the main adduct formed in cells treated with (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene.
Effects of Cereal Prolamin Peptides on Differentiated CaCo-2 Cells
Authors: Giovannini Claudio , Luchetti Roberto , Mancini Elena , de Vincenzi Massimo
Published: August 2020

The effects of peptic-tiyptic (PT) digests of prolamins derived from several cereals on differentiated CaCo-2 cells were studied on the nineteenth day of culture. Cell viability was determined by using the MTT assay and the colony-forming ability method. The metabolic consequences of peptide exposure were evaluated by measuring RNA, protein and glycoprotein synthesis. While PT digests from bovine serum albumin and durum wheat did not exert any effects, those derived from bread wheat, barley, rye and oats caused a dramatic inhibitory effect on metabolic synthesis and, when measured by using the colony-forming technique, a decrease in cell viability. The MTT assay did not indicate any changes in cell viability. These observations support the hypothesis that, although prolamin-derived peptides from these cereals do not exert an immediate cytotoxic effect, they are responsible for cell damage by impairment of metabolic processes.
Prooxidant Agents Induce Modifications in the Cytoskeletal Organisation of Fibroblasts
Authors: Miglietta Antonella , Gadoni Elena , Bocca Claudia , Gabriel Ludovica
Published: August 2020

An important modulatory role of free-radicals and related species in controlling the cell cycle has recently been revealed. The cytoskeletal system is an intracellular structure that participates in the regulatory mechanisms of the cell cycle and is involved in reactions which generate free-radicals. We studied the effects of three different prooxidant systems (ascorbate/iron II sulphate, adenosine diphosphate/iron III chloride and cumene hydroperoxide) on BALB/C 3T3 fibroblasts, in order to establish a relationship between cytoskeletal alterations and changes in cell proliferation under oxidative stress conditions. We found that prooxidant systems markedly influence cytoskeletal organisation, but only slightly modify the proliferation rate. The mechanism of cytoskeletal alteration is unclear, but does not depend on oxidative damage. The main components of the cytoskeleton appear to be involved in free-radical-generated cell reactions, even though a relationship with cell growth has yet to be identified.
Measurement of Xenobiotic Metabolising Enzyme Activities in Primary Monolayer Cultures of Immature Rainbow Trout Hepatocytes at Two Acclimatisation Temperatures
Authors: Jensen Elisabeth G. , Thauland Rune , Soli Nils E.
Published: August 2020

Rainbow trout hepatocytes with a high viability were isolated by two-step collagenase perfusion through the portal vein. The yield was 1-2.5 x 106 cells/g body weight. Culture conditions were defined for providing enhanced attachment and long-term cell survival at 7 ± 0.5°C and 15 ± 0.5°C, respectively. The hepatocytes, attached to Primaria™ plastic and cultured in Leibowitz L-15 medium with 9% fetal calf serum, were maintained as monolayers for 6–7 days. The activities in hepatocytes from immature trout of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD), aldrine epoxidase (AEPOX), NADPH-cytochrome c reductase (NCR), glutathione-S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT), were all stable during the culture period. Differences in enzyme stability and activity (particularly the activity of EROD) between hepatocytes from different fish were observed at both temperatures. The temperature did not influence the activities of EROD or NCR, whereas AEPOX showed metabolic compensation. Both GST and UDPGT exhibited inverse temperature compensation. Hepatocyte monolayers, cultured from immature trout, may provide a useful system in pharmacological and toxicological research for investigating drug metabolism.
Perspectives: Revisited
Authors: Kirkwood Hal
Published: August 2020

Perspectives: Revisited reviews a selection of articles across the SAGE journal list. This issue of the column focuses on business infromation resources, business strategy, gender diversity in business schools, business curriculum development, the United Nations Sustainable Develoment Goals, and the intersection of information literacy and management.
Preoperative C-reactive protein as a predictor of postoperative acute kidney injury in patients undergoing coronary artery bypass grafting
Authors: Mohamed Walid , Asimakopoulos George
Published: August 2020

C-reactive protein is a well-known marker of inflammation and may be preoperatively elevated in the absence of infection in adult cardiac surgery patients, indicating a baseline inflammatory process. We conducted a literature search to assess the available evidence on whether there is an association between preoperative C-reactive protein and acute kidney injury after coronary artery bypass grafting. Included only were observational studies which investigated this association. We excluded abstracts, case reports, animal studies and articles in languages other than English. Altogether, 199 papers were retrieved from the search strategy reported, of which 6 studies were included in the final review. The study types, publication details, patient groups and key results are tabulated. A qualitative assessment of these papers was conducted. We conclude that there is some evidence to support the association between high preoperative C-reactive protein level and postoperative acute kidney injury and complications after coronary artery bypass grafting. Owing to variability in acute kidney injury definitions and C-reactive protein levels for different cohorts in the observational studies reviewed, it is unclear from current evidence what serum C-reactive protein cut-off level is significantly associated with postoperative acute kidney injury. Further evidence is needed to investigate whether raised preoperative C-reactive protein in the absence of an infective cause could have a role in risk prediction models for cardiac surgery–associated acute kidney injury.