bluuebunny/crossref_metadata_2025 huggingface dataset:
Some leptothecate hydroids (Cnidaria, Hydrozoa) from Hawaii, mostly from inshore and nearshore waters
Authors: CALDER DALE R.
Published: August 2020

This report is based on a small collection of hydroids from the Hawaiian Islands, in the central Pacific Ocean. Most of the examined material was obtained by staff of the Bernice Pauahi Bishop Museum, Honolulu, during surveys for nonindigenous marine species in shallow, sheltered, inshore or nearshore waters, and especially in harbours, bays, and lagoons. In all, 34 species of leptothecate hydroids, assigned to 14 families and 20 genera, were identified and are discussed. One of them, based on a single infertile colony with a damaged hydrotheca, was identified provisionally only to the rank of suborder. Given the limited geographic and bathymetric focus of the surveys, only four of the species, Clytia thornelyi, Halecium sibogae, Macrorhynchia balei, and M. hawaiensis, were collected at depths greater than 25 m. Seven species, Cirrholovenia tetranema Kramp, 1959, Orthopyxis crenata (Hartlaub, 1901), Clytia elongata Marktanner-Turneretscher, 1890, C. paulensis (Vanhöffen, 1910), Tridentata maldivensis (Borradaile, 1905), Monotheca flexuosa (Bale, 1894), and a hydroid identified only as Eirenida (undetermined), are recorded from Hawaii for the first time. Three others, Lytocarpia nigra (Nutting, 1905) Macrorhynchia balei (Nutting, 1905), and M. hawaiensis (Nutting, 1905), have their type localities in Hawaii, with the last of these being known to date only from the Hawaiian archipelago. Most of the species are well-known from shallow water areas across the tropical and subtropical Indo-Pacific region, and over half of them have been reported as well from warm waters in the Atlantic Ocean. Their existence in the remote islands of Hawaii is attributed to long-range dispersal by both natural and human-mediated means, including shipping.
Assessing Risk in North Dakota Juvenile Probation: A Preliminary Examination of the Predictive Validity of the Youth Assessment and Screening Instrument
Authors: Matz Adam K. , Martinez Adrian R. , Kujava Elizabeth
Published: August 2020

This exploratory research examines the predictive accuracy of the Youth Assessment and Screening Instrument adopted by the North Dakota Juvenile Court through a retrospective review of assessment and court records. While studies of YASI from New York, Virginia, and Canada provide some confidence in the instrument’s predictive validity, questions remain concerning its accuracy among female and other specialized populations. This study finds a moderate effect for the instrument’s predictive accuracy in relation to general reoffending from a random sample of juvenile probationers (AUC = 0.66, p = .002, 95% CI [0.56, 0.75], N = 139), but results were notably weaker for females compared to males. Further research is needed on its accuracy among African American and Native American youth.
Sexual health among Kenyan male sex workers in a time of COVID-19
Authors: Macharia Pascal , Moore Samantha , Mathenge John , Ndunda Erastus , Lazarus Lisa , McKinnon Lyle R , Lorway Robert
Published: August 2020

Objective:

This report identifies the profound effects that the COVID-19 pandemic and the resultant government lockdown have had on sexual health services delivery to a community of marginalised male sex workers in Nairobi, Kenya.

Methods:

Based on the experiences shared during ongoing virtual conversations with peer health workers, a case study was developed to identify the challenges encountered by peer health workers.

Findings:

Peer health workers confronted the new health crisis surrounding COVID-19 while also persisting in their efforts to deliver HIV services to male sex workers. Unable to receive status as ‘essential workers’, their actions often fell short in efforts to maintain male sex workers’ access to vital HIV prevention, treatment and care resources.

Conclusion:

The struggles encountered, amid dwindling resources, underscore the vital work needed to meet the health needs of a marginalised group that remains largely excluded from the government health system.
Evaluation of mechanical properties of fiber reinforced composites filled with hollow spheres: A micromechanics approach
Authors: Biarjemandi Mojde , Etemadi Ehsan , Lezgy-Nazargah Mojtaba
Published: August 2020

Recent researches show that the embedment of hollow spheres in the matrix phase of composite materials improves the strength of these structures against crack propagations. Rare studies are reported for calculating equivalent elastic constants of fiber reinforced composites containing hollow spheres. In this paper, the effects of hollow spheres on mechanical characteristics of fiber reinforced composite are studied for the first time. To achieve this aim, a micromechanics based finite element method is employed. Representative volume elements (RVEs) including hollow spheres with different radius, thickness and volume fraction of hollow spheres, are modeled by using 3D finite elements. The equivalent elastic constants are calculated through homogenization technique. The results are compared with available experimental works. Good agreements find between two sets of results. Also, the volume fraction, number and thickness of hollow spheres as effective parameters on mechanical properties of composite were investigated. The results show the equivalent elastic properties increase with increasing the volume fraction and number of hollow spheres and decrease with increasing the number of hollow spheres. Furthermore, the equivalent Young’s modulus in transverse directions to the fiber direction and shear modulus of the composite increase with increasing the thickness of hollow spheres. As a final result, the presence of hollow spheres in the matrix phase generally increases the equivalent elastic constants without significant changes in the weight of structures.
Gendered Boundary-work within the Finnish Skepticism Movement
Authors: Vuolanto Pia , Kolehmainen Marjo
Published: August 2020

As a worldwide social movement, skepticism aims to promote science and critical thinking. However, by analyzing texts published in the magazine of the Finnish skepticism movement between 1988 and 2017, we find that the movement carries out its mission in a way that maintains and produces gendered hierarchies. We identify six forms of gendered boundary-work in the data: (1) science as masculine, (2) questioning women, (3) complementary and alternative medicine as feminine, (4) debating the status of gender studies, (5) gender within the skepticism movement, and (6) supporting equality. Gender is an important aspect of the boundary-work undertaken by the movement to establish boundaries between science and nonscience. The forms of gendered boundary-work contribute to the idea of “true” science as a masculine and male-dominated domain, excluding women from both science and the skepticism movement. Even when the exclusions are subtle, hidden, or humorous, they nevertheless produce gendered inequalities by excluding women, belittling women’s knowledge production, or granting women-only dismissive recognition. Indeed, our analysis indicates that there is a need to look deeply into science-based social movements: exclusive structural tactics are part and parcel of such movements’ mundane activities, as our examples from Skepsis ry’s popular magazine demonstrate.
Warfarin-induced toxic epidermal necrolysis after mitral valve replacement
Authors: Kuroda Taiyo , Yokoyama Yukifusa , Yamada Masao , Yuhara Satoshi , Hasegawa Hiroki , Yokote Jun
Published: August 2020

Stevens-Johnson syndrome and toxic epidermal necrolysis are rare diseases that cause acute destruction of the epithelium of the skin and mucous membranes, almost always attributable to drugs. However, warfarin-induced Stevens-Johnson syndrome and toxic epidermal necrolysis is extremely rare. We report the case of 71-year-old woman who died due to destructive erosion all over her skin and mucous membranes. She had received a mitral valve prosthesis, and warfarin was prescribed for antithrombotic therapy. A lymphocyte transformation test for drug hypersensitivity and the clinical history confirmed this phenomenon as warfarin-induced toxic epidermal necrolysis.
Microcomputer Simulations of Mechanical Properties of Skeletal Muscle for Undergraduate Classes
Authors: Clarke Kenneth A.
Published: August 2020

In a previous paper, the development of interactive simulations of neurophysiology experiments for undergraduate classes was described. Similar voltage/time displays are used in other areas of undergraduate physiology classes and could therefore be simulated in this way. One such area is in the study of the mechanical properties of muscle, in which a transducer is used to translate muscle length or tension changes into voltages. This paper describes the development and class use of interactive simulations of experiments using isometric recordings from isolated frog muscle.
Acrylamide, 2,5-Hexanedione and β-Aminopropionitrile Toxicity Tested in Rat Embryo Mid-brain Cell Cultures
Authors: Walum Erik , Flint Oliver
Published: August 2020

The abilities of three substances, acrylamide, 2,5-hexanedione and β-aminopropionitrile to affect nerve cell differentiation were compared with their general cytotoxicities (ability to affect cell survival) over five days in rat embryo mid-brain micromass cultures. ACA and 25HD are neurotoxins in the adult, but only ACA is known to have transplacental effects on the developing central nervous system. BAPN is a model teratogen, but not a neurotoxin, in the adult and was used as a positive control. ACA inhibited neuronal differentiation (assayed by reduction of number of foci of differentiated neurons) by 50% at a concentration (the IC50) of 15μg/ml. The corresponding IC50 for cell survival (assayed by reduction in cell number) was 36μg/ml. In 25HD-exposed cultures, IC50 values of 729 and 872μg/ml were obtained for the inhibition of differentiation and cell survival, respectively. BAPN inhibited differentiation with an IC50 of 226μg/ml, but had very little effect on cell viability up to 500μg/ml. This pronounced difference in sensitivity of the two endpoints is in agreement with previous reports of its teratogenicity. The more-limited but significant difference detected for ACA, but not for 25HD, suggests that rat embryo mid-brain micromass cultures can be used to study subteratogenic effects of ACA on brain development.
Metal Toxicity and Metallothionein mRNA Induction in a Rat Intestinal Cell Line
Authors: Scarino Maria L. , Bises Giovanna , Leomporra Lucrezia , Zucco Flavia
Published: August 2020

Our aim was to characterise a rat intestinal cell line, IEC-17, isolated by Quaroni from neonatal rat duodenum, as an in vitro model for the study of metal metabolism and toxicity. Dose-effect experiments were carried out using two essential trace elements (zinc and copper) and one toxic metal (cadmium). The results showed different trends of responses between physiological and toxic metals; the LD50 values were 250μM for Zn, 600μM for Cu, and 5μM for Cd. Since metallothioneins are the only identified and characterised proteins involved in metal detoxification, we tried to establish a relationship between metallothionein gene expression and resistance to metal toxicity. Metallothionein in mRNA induction was studied by the Northern blot technique, using an MT-1 cDNA probe. A basal level of metallothionein mRNA was detectable in cells growing in normal medium. Induction by Cd of metallothionein mRNA was very strong after 6 hours. Both Zn and Cu were also able to induce MT mRNA after 6 hours in IEC-17 cells in a dose-dependent manner.
Toxicity of Fenclor 42 on Immunocompetent Cells
Authors: Franco Paola , Marelli Ornella , Canti Gianfranco , Ricci Laura , Prandoni Nancy
Published: August 2020

Polychlorinated biphenyls (PCBs) are environmental contaminants, whose toxicity is related to the degree and the position of chlorine substitutes. Fenclor 42 is a mixture of trichlorobiphenyls employed for industrial use in Italy. In order to evaluate its effects on the immune system, CD2F1 mice were treated with Fenclor 42 at different doses and schedules. Spleen weights, total number of splenocytes and relative spleen weight decreased significatively following a single or a prolonged exposure to different doses of Fenclor 42.

An analysis of the functional activity of splenocytes showed that the proliferative response to Con A and PWM mitogens was also inhibited by Fenclor 42. Splenic parameters returned to normal values within 5 days after a single injection and between 8–15 days after a subchronic administration of the compound. The capability of splenocytes to proliferate, following mitogen stimulation, was restored between day 5 and day 8 according to the different schedules of treatment. By comparison, Fenclor 42 never affected NK activity.

These data clearly indicate damage at the splenic level by Fenclor 42. Studies are in progress to elucidate the mechanisms involved in the immunotoxic response.
In Vitro Cytotoxicity of 150 Chemicals to 3T3-L1 Cells, Assessed by the FRAME Kenacid Blue Method
Authors: Clothier Richard H. , Hulme Lesley , Ahmed Aamer B. , Reeves Helen L. , Smith Moira , Balls Michael
Published: August 2020

The cytotoxicities to 3T3-L1 cells of 150 coded chemicals, estimated by the FRAME kenacid blue method, are given as a basis for further studies on the relative in vitro toxicities of related and distinct sets of test chemicals, and as a basis for future attempts at the comparison and correlation of estimates of toxicity obtained in vitro and in vivo.

The expression of the results of in vitro cytotoxicity tests is discussed, as are various ways of comparing values obtained for different chemicals. It is suggested that there should be agreement on the inclusion of a standard set of reference chemicals in all cytotoxicity test validation trials.
Laboratory Animals in Bacterial Vaccine Control and Some Opportunities for Replacement, Reduction and Refinement
Authors: Hendriksen Coenraad F.M.
Published: August 2020

In recent years, there has been a growing interest in alternatives to animal experimentation. Public attention was, and still is, focused on potential alternatives in toxicology in particular, probably because of the moral implications of testing some chemical products (e.g. cosmetics) by means of in vivo procedures such as the Draize eye test. In contrast, the use of laboratory animals for quality control of biological products such as vaccines is scarcely criticised. Nevertheless, the use of animals for that purpose is extensive and alternatives are urgently needed for several reasons. The development of models which can reduce the number of animals used or refine the experimental procedures used is receiving attention at several institutions. This paper outlines the activities of the Laboratory for the Control of Bacterial Vaccines of the National Institute of Public Health and Environmental Protection (RIVM) in the Netherlands, especially with regard to the testing of diphtheria and tetanus vaccines.
Induction of Porphyrin Biosynthesis in the Non-Animal Experimental Model of Tissue Explant Cultures: Prevention and Reversal by the Antimitotic Colchicine
Authors: Buzaleh Ana Maria , Afonso Susana G. , Polo Cesar F. , Navone Nora M. , Bianchi Alberto , Schoua Ernesto , Del C. Batlle Alcira M. , Vazquez Elba S.
Published: August 2020

The addition of different concentrations (5–20mg/dish) of allyl isopropylacetamide (AIA) and veronal (VER) to mouse liver, skin and heart tissue explant cultures increased the biosynthesis of porphyrin from endogenous precursors several-fold as compared to controls.

Maximal effects were observed after a 12-hour incubation in liver culture for both AIA and VER (at 5mg drug/dish); after 2 and 6 hours in skin for AIA (10mg/dish) and VER (5mg/dish). In heart, with AIA (5mg/dish) and VER (20mg/dish), a peak was found after 2 hours, but a second increase was observed later after AIA induction.

Colchicine (O.1mg/dish) did not by itself alter porphyrin biosynthesis by any tissue However, when added before or after porphyrin induction by AIA, it was able to both prevent and reverse the effect of the inducer in liver explants only, having no action whatever on other tissues or when VER was used.

The usefulness of the non-animal experimental model of tissue explant cultures for investigating the porphyrinogenic properties and/or toxicity of drugs and their mechanism of action is emphasised.
Toxicological Responses to Zinc, Copper and Cadmium in a Rat Intestinal Cell Line
Authors: Bises Giovanna , Ranaldi Giulia , Scarino Maria Laura , Zucco Flavia
Published: August 2020

An intestinal epithelial cell line from neonatal rat duodenum (IEC-17) was used as a model for investigating metal toxicity.

In this paper, we have further characterised toxic effects on IEC-17 cells following exposure to two physiological metals, zinc (Zn2+) and copper (Cu2+) and one non-physiological metal, cadmium (Cd2+). Time-effect experiments showed that the duration of the exposure affected the extent of cell damage only when Cu2+ and Cd2+ were used. During the first 48 hours of Zn2+ exposure, the cells were seriously affected, but subsequently were able to recover. On the other hand, a colony forming ability test and morphological observations showed a special sensitivity of this cell line to Cu2+. A possible explanation is suggested in relation to extracellular matrix formation.
Cytotoxic and Genotoxic Effects of 10 N-Nitroso Compounds in a Human Hepatoma Cell Line (Hep G2): Comparison with Human Hepatocyte Primary Cultures
Authors: Campart Giulia Brambilla , Canonero Roberta , Mereto Eugenio , Ferro Margherita
Published: August 2020

A comparison of data from assays performed in cells of an established line derived from a human hepatoblastoma (Hep G2) with those previously obtained in freshly-isolated human hepatocytes, reveals that the latter are, by varying degrees, more sensitive than the former to the cytotoxic and genotoxic effects of 10 N-nitroso compounds, independently of their nature as proximate or ultimate carcinogens.
A Case Study on the Impact of Clinically-Observed Abnormalities in Mice with Gallstones on the Ethical Admissibility of a Projected Experiment with Gallstone-Bearing Mice
Authors: Stafleu Frans R. , Heeger F. Robert , Beynen Anton C.
Published: August 2020

An attempt was made to study the impact of clinically-observed abnormalities in mice with gallstones on the ethical admissibility of a proposed experiment using gallstone-bearing mice. Three groups of respondents completed a questionnaire based on a hypothetical protocol of a proposed experiment using gallstone-bearing mice. Differing information was given in the protocols concerning abnormalities in gallstone-bearing mice. Respondents were asked to judge, using a scoring system, the ethical admissibility of the proposed experiment and the degree of discomfort for the mice. Respondents were also asked to defend their admissibility judgements and discomfort scores. Respondents given clinical information considered the degree of discomfort to be more serious than respondents without such information. Respondents given information indicated that their estimate of discomfort was based principally on the clinical information provided. Respondents without clinical information mentioned the lack of such information as a handicap to assessing discomfort properly. Scores on ethical admissibility of the proposed experiment were found to be unaltered by the clinical information. Judgement of the ethical admissibility was based mainly on the anticipated human benefits to be gained by performing the experiment. It is stressed that the specific character of the present study does not allow generalisation of its outcome.
A Computer Simulation of the Cat Nictitating Membrane Preparation (In Vivo) for Teaching Undergraduate Pharmacology Students
Authors: Dewhurst David G. , Howells Clint M.
Published: August 2020

An interactive computer-assisted learning program is described, aimed at teaching students, on a range of undergraduate/diploma level courses, the basic pharmacology of transmission at autonomic ganglia and sympathetically innervated smooth muscle. The program, which is menu-driven, simulates experiments which can be conducted on the superior cervical ganglion nictitating membrane preparation (in vivo) of the anaesthetised cat, and is written for IBM-compatible microcomputers. Students “design” experiments by selecting certain experimental parameters such as: an appropriate drug to investigate from a comprehensive list; the site of administration; and whether to stimulate pre-ganglionic nerves. Simulated contractions of the nictitating membrane, derived from actual experimental data, are presented on a scrolling display on the monitor in a form comparable to that of a chart recorder. The learning package includes background information and teacher-designed help screens, and is supported by tutors’ notes and suggested assignments. The use of the program in teaching pharmacology and as an alternative to animal experiments, is discussed.
Development of Potential Alternatives to the Draize Eye Test: The CTFA Evaluation of Alternatives Program
Authors: Gettings Stephen D. , McEwen Gerald N.
Published: August 2020

The CTFA Evaluation of Alternatives Program is a multi-year effort organised by a scientific advisory committee (the CTFA Animal Welfare Task Force), and is designed to evaluate (in Phase I) approximately twenty-five potential alternative methods to the Draize eye irritancy test. Coordination, management of logistics, collection and statistical analysis of data, are being conducted by the Columbus Division of the Battelle Memorial Institute. The US Food and Drug Administration has been aware of the Program since its inception. The intention of the Program is to provide industry with information on the performance of a series of potential alternatives to the Draize test, so as to aid individual companies to identify those methods which seem best suited to their own particular testing needs. The participants are either CTFA member companies who are already using or developing alternative tests, or those independent investigators whose development work is being sponsored by CTFA members. The purpose of the Program is to determine the effectiveness and limitations of a variety of tests for a range of different product types. Specifically, the Program will evaluate the capacity of the tests under investigation to rank and discriminate between the ocular irritation potential of a range of prototype cosmetic and personal care products. The Program is designed as a multi-year effort with a different product type evaluated each year. In Phase I, ten different ethanol-based substances are being tested; oil/water emulsions will be evaluated in Phase II.
In Vitro Toxicity Testing: Purpose, Validation and Strategy
Authors: Flint Oliver P.
Published: August 2020

The fullest potential for in vitro evaluation of toxicity will be realised in the context of the process of assessing the risk of human toxicity. This article is an attempt to clarify what contributions can be made by in vitro tests and what types of in vitro test can best be used. In vitro tests are clarified according to the type of biological endpoint evaluated, first into tests for general (‘basal’) cytotoxicity and, secondly, into tests for differentiated cell function. The role of each type of test is analysed and it is suggested that tests for general cytotoxicity, as opposed to differentiated function, are difficult to interpret in terms of in vivo toxicity. A general approach to evaluating in vitro tests is described, and a strategy for using these tests is proposed.
Adverse Effects in Animals and their Relevance to Refining Scientific Procedures
Authors: Morton David B.
Published: August 2020

This paper highlights the areas in which refinement, with the specific aim of reducing laboratory animal pain, distress and anxiety, can be achieved. Good husbandry and housing which meet the animals’ behavioural needs, careful and gentle handling, competence in carrying out scientific procedures, and alleviation of any unwanted side-effects, are all of paramount importance. Whilst “suffering” cannot easily be tightly defined, it is essential to recognise when an animal is suffering so that its alleviation can be instigated.
Reduction of Animal Experiments in Education in the Netherlands
Authors: Nab Jan
Published: August 2020

From an academic point of view the use of animals in education is primarily as tools to achieve learning objectives. The introduction of alternatives in education requires an appreciation of both the learning objectives of these animal experiments and the possibilities and limitations of alternatives. In the Netherlands, a government-funded project is in progress, with the aim of developing the use of alternatives to animal experiments in education The numbers of animals used for educational purposes was reduced by 40% in the period 1978–1988

This paper presents an overview of the objectives of the use of animals for experiments in education and of the alternatives currently available to academia.
A Decade of European Legislation — Can we Legislate for Humanity?
Authors: Hampson Judith E.
Published: August 2020

The establishment throughout Europe, during the last decade, of legislation to control animal experiments, is described. The effectiveness of such laws to achieve their objectives is discussed. In particular, the effectiveness of costs against benefit as a part of project authorisation, is considered.
Information Systems and Databases as Alternatives
Authors: Bawden David
Published: August 2020

The influence of information technology on the development of computerised information systems which can contribute to reduction and replacement of animal experimentation is considered. Particular emphasis is placed on access to information from computer databases as an alternative in itself. The role of databases in support of modelling (molecular and statistical), simulation and risk assessment is also described.
The Safety Evaluation of Drugs and Chemicals by the Use of Computer Optimised Molecular Parametric Analysis of Chemical Toxicity (COMPACT)
Authors: Parke Dennis V. , Ioannides Costas , Lewis David F.V.
Published: August 2020

The historical development of the safety evaluation of drugs and chemicals is critically reviewed, and failures of the present approach using experimental animals are examined. The roles of the cytochromes P450 in the detoxication of drugs and chemicals, and in their activation to mutagens, carcinogens and neoantigens, are described, and the importance of the selective induction of the cytochromes P450 in the manifestation of chemical toxicity/carcinogenicity is highlighted. The computer graphic procedure of COMPACT, which relates chemical structures to metabolism by individual cytochromes P450, and hence to their potential toxicity/carcinogenicity, is described, and the advantages and disadvantages of this method of safety evaluation, which does not use experimental animals, are considered.
Loss of Trans-epithelial Impermeability of a Confluent Monolayer of Madin-Darby Canine Kidney (MDCK) Cells as a Determinant of Ocular Irritancy Potential
Authors: Shaw Andrew J. , Clothier Richard H. , Balls Michael
Published: August 2020

The short-term exposure of a confluent layer of MDCK cells to a series of test agents of varying ocular irritancy potential demonstrated that the tight junctions between these cells were affected in such a way that fluorescein could leak through in a dose-related manner. Rankings of test agents in order of potency to promote “fluorescein leakage” correlated well with neutral red release results (r=0.98) and ranked toxicity closely with in vivo eye irritancy data. However, the neutral red release assay produced negative results for three inorganic irritants, whereas these were detected using the fluorescein leakage assay.
Studies of Metabolism Mediated Mutagenicity In Vitro
Authors: Jenssen Dag , Romert Lennart
Published: August 2020

To understand the cause of the biological effects of xenobiotic metabolism in mammals, investigators have traditionally performed animal experiments by comparing the results of biochemical methods, such as measurement of enzyme activity analysis of the metabolites produced, with the observed toxicological effect. This article deals with in vitro methods for genotoxicity combined with drug metabolising preparations at the organelle, cell or organ levels, as exemplified by microsome preparations, isolated cells/cell lines and organ perfusion systems, respectively.

The advantage of some of these methods for studying metabolism-mediated mutagenicity is that the measured endpoint reflects not only the bioactivating phase I reactions, but also the detoxifying phase II reactions, and the transfer of the non-conjugated reactive metabolites to other cells and their ability to cause mutations in these cells. In vivo, all these events are important factors in the initiation of cancer. A mechanistic advantage of the methods for metabolism-mediated mutagenicity in vitro is that the relevance of the different steps in metabolism for the mutational events can seldom be investigated in an in vivo assay. Furthermore, human studies can easily be performed using the co-culture technique with isolated human cells or cell lines.
In Vitro Models to Study Mechanisms of Lung Toxicity
Authors: Ryrfeldt Åke , Cotgreave Ian A. , Moldéus Peter
Published: August 2020

Several functioning in vitro systems of varying complexity are currently in use for the study of mechanisms of lung toxicity. The isolated perfused lung is the model closest to the in vivo situation. It is a suitable model for combining metabolic and functional studies. It is, for instance, possible to relate changes in lung mechanics and lung perfusion flow to the release of various mediators during exposure of the lung to various agents. A simpler model may be constructed from lung slices which are less viable but suitable for uptake as well as metabolism studies.

Specific lung cells such as Clara cells and type II pneumocytes have been isolated and cultured and are valuable tools for studies of the molecular mechanisms of lung toxicity, particularly in cases of cell-specific toxicity. There is, however, a great need to develop techniques for the isolation and culture of other types of lung cells and also to improve the culturing techniques for those already isolated.
Spectrophotometric Determination of Total Macromolecules for the Evaluation of Cellular Density in Cytotoxicity Tests
Authors: Fortunati Elisabetta , Bianchi Vera
Published: August 2020

Variation of cell number in treated cultures relative to the controls is an indicator of toxicity frequently employed in cytotoxicity tests. Cell density is generally determined from the amount of macromolecular components (DNA, proteins) measured by colorimetric methods. Another established procedure is the neutral red (NR) uptake test, which, in principle, permits an evaluation of the amount of viable cells in the different samples. We have devised a simple method for comparing cell density in the cultures, based on the spectrophotometric determination of the total macromolecules present in the cell monolayers solubilised in alkali after removal of the soluble fraction (TM test). When applied in parallel with the NR assay in a cell viability assay, our TM test gives a concordant rank of toxic potency for the chemicals tested. In comparison with NR uptake, the TM method appears to be more accurate and the data it provides are less prone to variation.
Toxicity of Ethylene-bis-dithiocarbamates (EBDCs) in a Human Neuroblastoma Cell Line
Authors: Cova Dario , Fumagalli Pietro , Santagostino Angela
Published: August 2020

The aim of our research was the in vitro evaluation of the neurotoxic effects of three EBDCs (Nabam, Zineb and Maneb) and ETU on SK-N-BE human neuroblastoma cells as a model for neurotoxicity in humans.

The EC50 value was used as an index of the toxicities of these compounds. Since Zineb and Maneb contain zinc and manganese as cations, respectively, in order to determine the contributions of these metals, the EC50s of zinc chloride and manganese chloride were also evaluated.

Nabam, Zineb and Maneb had EC50 values ranging from 1μM to 30μM; the EC50s of manganese and zinc in this human cell line were found to be of the same order of magnitude as those of the EBDC fungicides. These in vitro effects are discussed in relation to the possible use of neuronal cell lines for detecting the neurotoxicities of these compounds.
An In Vitro Model for Studying Oxidative Damage and Protective Substances in Human Cells
Authors: Monti Daniela , Barozzi Daniela , Buttafoco Paola , Troiano Leonarda , Tropea Franco , Cossarizza Andrea , Grassilli Emanuela , Pelioni Maria Claudia , Marini Marina , Tiozzo Roberta , Franceschi Claudio
Published: August 2020

An in vitro experimental system comprising human peripheral blood lymphocytes or rat splenocytes exposed to oxygen free radicals (OFR) generated extracellularly by a classic enzymatic system, i.e. xanthine oxidase (XOD) plus hypoxanthine (HYP), is proposed as a model for the study of OFR-induced damage, for investigating the mechanisms responsible for cell injury and for identifying possible protective substances. We show that rat splenocytes are much more sensitive than human lymphocytes to oxidative stress. The protective effects exerted on OFR-exposed human lymphocytes by inhibitors of poly(ADP-ribosyl)polymerase (ADPRP), such as 3-aminobenzamide, 3-methoxybenzamide and nicotinamide, suggest that the activation of ADPRP is a critical step in the metabolic pathways which mediate the toxic effect of OFR in human cells.
Oxidant-induced Inhibition of DNA Synthesis
Authors: Sestili Piero , Fiorani Mara , Cattabeni Flaminio , Cantoni Orazio
Published: August 2020

The DNA synthesis inhibitory effect of hydrogen peroxide is more pronounced when treatments are performed at 37°C than at 4°C and in low density rather than high density cultures. Recovery from DNA synthesis inhibition appears to be rapid in cells challenged with the oxidant at 4°C, whereas no recovery (for up to 6 hours) was apparent in cells treated at 37°C. These results correlate well with our previous data, indicating that different types of lesions are produced by hydrogen peroxide at the two different temperatures, and suggest an explanation for the lower toxicity elicited by the oxidant at 4°C, as compared to the physiological temperature of 37°C.
Effects of Neutral Shampoos on Cultures of Human Fibroblasts
Authors: Cavalletti Teresa , Reali Eva , Marangoni Claudia , Bader Stefano , Traniello Serena
Published: August 2020

Criteria used to predict the effect of neutral shampoos on fibroblast cultures were based on assays which were easy to perform, reproducible and adaptable to a large number of substances. The tests were based on the determination of the integrity of the plasma membrane, the rate of protein synthesis, and cell viability.

Six different shampoos were tested: four were of the best quality commercially available, while the other two were new compounds. The substances were tested over a wide concentration range and were applied to human fibroblast cultures for various exposure times. Lactate dehydrogenase release into the medium was used as a marker of plasma membrane integrity, the amount of [3H]-labelled protein in the fibroblasts was measured to assess the function of the cell biosynthetic machinery, and the number of damaged cells which did not take up the neutral red dye showed the percentage of cytolysis.

The results indicated significant differences between the six shampoos, and a relationship between the biochemical parameters was found. This suggests that these tests on cell functionality may represent reliable markers for monitoring the cellular target damaged by xenobiotic compounds.
Teratogenicity Testing In Vitro: Post-implantation Whole-embryo Culture
Authors: Giavini Erminio , Broccia Maria Luisa , Prati Mariangela
Published: August 2020

Various in vitro models have recently been tested for the assessment of developmental toxicity. Since the mechanisms involved in chemically-induced embryotoxicity are complex and numerous, a good in vitro assay must be able to detect developmental toxicants which act via most or all of these mechanisms. The rodent whole-embryo culture seems to fit this requirement, because it undergoes all of the fundamental processes of development. Also physical relationships between cells and tissues are maintained and morphogenesis can proceed normally. In fact, in vitro development of early post-implantation embryos closely parallels that occurring in utero. This assay appears to be particulary relevant for the detection of teratogenic and embryotoxic chemicals.
Polyclonal IgY Antibodies from Chicken Egg Yolk — an Alternative to the Production of Mammalian IgG Type Antibodies in Rabbits
Authors: Schade Rüdiger , Pfister Claus , Halatsch Rainer , Henklein Peter
Published: August 2020

Based on a physiological process, antibodies are concentrated in the yolk of chicken eggs, and can be used as a convenient source of polyclonal antibodies. Hens were immunised with a BSA-cholecystokinin octapeptide conjugate (CCK-8) to obtain antibodies against CCK-8. A simple method is described for extracting the antibodies from egg yolk, which can then successfully be used in immunocytochemical studies. Using these antibodies, CCK-like immunoreactivity was found in brain sections of rats, both by fluorescein isothiocyanate and peroxidase staining. The specificities of these antibodies were different from those of rabbit antibodies against the C-terminal part of CCK-8.

The applicability of egg yolk antibodies in immunological investigations is reviewed and discussed in detail. The properties of avian egg yolk antibodies are compared with those of polyclonal antibodies obtained from mammals (e.g. the rabbit). The more general use of avian antibodies is emphasised as a real alternative, taking into consideration the reduction in animal suffering involved in comparison with the classical immunisation techniques used on rabbits.
Chemical Contamination Suspected in Cytotoxic Effects of Incubators on BALB 3T3 Cells
Authors: Sasaki Kiyoshi , Tanaka Noriho
Published: August 2020

We investigated the cytotoxic effects of CO2 incubators manufactured by company A on the growth of three cell lines, BALB 3T3, L-929 and V79. Proliferation of BALB 3T3 and L-929 cells was markedly inhibited, although BALB 3T3 were more highly susceptible than L-929, and cell growth decreased with decreasing cell density: no colonies were formed when 100 BALB 3T3 or L-929 cells per dish were plated. The growth of V79 cells, however, was not affected. Further, a drastic reduction in colony formation was observed when BALB 3T3 cells were plated in media that had been preincubated in an incubator from company A, then cultured in a different incubator that had previously been shown to have no such cytotoxic effects. All the incubators used in this investigation were shown to be mechanically and electronically in good condition, and to be operating perfectly, being able to maintain temperature, humidity and CO2 concentration satisfactorily. This study suggests that the cytotoxic effect of company A's incubators is being caused by chemical contamination from materials used during manufacture.
Hepatocytes in Primary Culture: An Alternative to LD50 Testing? Validation of a Predictive Model by Multivariate Analysis
Authors: Peloux Anne-Françoise , Fédérici Christian , Bichet Nicole , Gouy Daniel , Cano Jean-Paul
Published: August 2020

The cytotoxicity of 30 chemicals was assessed in rat hepatocyte primary cultures using four methods: lactate dehydrogenase release, neutral red uptake, the MTT assay, and measurement of total protein content.

Comparison of the data obtained in vitro (IC50 values) and in vivo (LD50 values) resulted in a significant correlation (p<0.001) between IC50 values and intravenous LD50 values. The validity, as well as the predictability of the model, were determined by multivariate analysis (principal component analysis and correspondence analysis). The predictability area, expressed in IC50 values, was in the range of 0–l,500μg/ml and reached 95%, with a 75–100% confidence interval (p = 0.05).

Assessment of the cytotoxicity of 54 additional chemicals would provide a more accurate predictability limit around l,500μg/ml and the estimated predictability confidence interval could be reduced to 90–100%.
Functional Alterations of Endothelial Cells Cultured In Vitro in Contact with Biomaterials
Authors: Cenni Elisabetta , Ciapetti Gabriela , Stea Susanna , Di Leo Alessandro , Cavedagna Daniela , Pizzoferrato Arturo
Published: August 2020

In order to evaluate in vitro the suitability of various prosthetic materials for endothelial seeding, human endothelial cells derived from the umbilical vein were placed in direct contact with a variety of polymers. As a control, endothelial cells were cultured in the absence of material. After 24, 48, 72 and 96 hours, the cells were counted and a viability test with neutral red was performed. Assays of 6-keto prostaglandinFla (6-keto-PGF1a), tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) were carried out on the supernatants. The cell counting technique demonstrated growth inhibition in the cell populations in contact with Woven Dacron® and Double Velour Dacron® in comparison with control cell cultures. Vital staining with neutral red, always sharply positive in the controls, was weak in the cells placed in contact with the materials. The 6-keto-PGF1a concentration in the supernatant was similar to the control level in the populations in contact with Woven Dacron® and Double Velour Dacron®. The tPA synthesis was higher in the cells exposed to the assayed materials compared to control values, while the PAI-1 concentration in the supernatant was lower in the cultures in contact with all materials.
Renal Cortical Slices: An In Vitro Model for Kidney Metabolism and Toxicity
Authors: Trevisan Andrea , Maso Stefano , Meneghetti Paola
Published: August 2020

The in vitro renal cortical slice model was used to study: 1) the effects on the kidney of some haloalkanes and haloalkenes using 3-month-old male Wistar rats; 2) influence of age and sex on renal cortical slice indices in non-treated rats; and 3) effects of 1,2-dichloropropane on the slices after pretreatment of 3-month-old male Wistar rats with DL-butathionine-[S,R]-sulphoximine. The most nephrotoxic chemical used was 1,3-dichloropropene, which caused a total depletion in the levels of reduced glutathione, a high peroxidation of lipid (about three thousand-fold with respect to control), a significant release of tubular enzymes into the medium, and loss of organic anion ( p-aminohippurate) accumulation. All the chemicals affected the cytosol more than the brush border. The most remarkable age-related differences in the untreated slices were the progressive decrease of reduced glutathione (p<0.05 from three months of age), and an increase in lactate dehydrogenase release into the medium (p<0.05 from six months of age). By contrast, sex differences were slight. The ‘treatment with 1,2-dichloropropane of slices prepared from rats pretreated with DL-butathionine-[S,R]-sulphoximine significantly increased the depletion of glutathione content (p<0.05) and malondialdehyde release in the medium (p<0.001) caused by the solvent alone.
Evaluation of Toxicity Testing of 20 MEIC Chemicals on Hep-2 Cells Using Two Viability Endpoints
Authors: Zanetti Cristiana , De Angelis Isabella , Stammati Anna-Laura , Zucco Flavia
Published: August 2020

The aim of the MEIC programme is to validate different in vitro testing protocols at an international level, comparing the results obtained for a series of compounds with the respective human in vivo data.

The first 20 chemicals of the MEIC programme have been tested on a human epithelial cell line, Hep-2. Dose-response curves were determined by two viability endpoints, total protein content and percentage of LDH released, after exposure of confluent cultures for 24 hours. A good agreement was found in the LC50 ranking of the compounds obtained by the two parameters. Moreover, the effects of these compounds on the energetic pathway have been studied using a Clark electrode, at doses corresponding to the LC50 and just after the addition of the compound to the cells. A remarkable difference in the overall picture of toxicity is detected by assessing this specific target.

The data obtained in this study are in good agreement with other, already published, MEIC project results.
Development of Potential Alternatives to the Draize Eye Test: The CTFA Evaluation of Alternatives Program. Phase II: Review of Materials and Methods
Authors: Gettings Stephen D. , Bagley Daniel M. , Chudkowski Michael , Demetrullas Janis L. , DiPasquale Louis C. , Galli Corrado L. , Gay Roger , Hintze Kenneth L. , Janus Jeffrey , Marenus Kenneth D. , Muscatiello Michael J. , Pape Wolfgang J.W. , Renskers Kevin J. , Roddy Martin T. , Schnetzinger Richard
Published: August 2020

The CTFA Evaluation of Alternatives Program is a multi-year effort, organised by the CTFA Animal Welfare Task Force, designed to evaluate the performance of currently promising in vitro (alternative) methods to the Draize eye irritancy test. The sole criterion for inclusion of a particular test is that it shows some initial promise as an alternative to the Draize eye test, and that it is under evaluation or development by a participating CTFA member company. Tests are evaluated for their ability to rank and discriminate the ocular irritation potential of prototype cosmetic and personal care formulations compared to the Draize eye test. Test materials and in vitro methods currently under evaluation in Phase II of the CTFA Program are described. Additional tests may be included in subsequent phases of the Program, should it be determined that they show particular promise as replacements for specific types of formulation. Conversely (at the discretion of sponsors), tests may be removed from the Program should initial promise be unfulfilled.
Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy
Authors: Bjerregaard Henning F.
Published: August 2020

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.