ATLA 25.3, May 1997

//ATLA 25.3, May 1997

Non-animal Alternatives and Reduction in Laboratory Animal Use

Michael Balls & Julia Fentem

The Three Rs of Russell & Burch represent not only a unifying concept, but also a unified concept. There are many circumstances in which we cannot separate reduction, refinement and replacement. The development and application of non-animal procedures for use in the biomedical sciences is one such circumstance. Such replacement alternatives are contributing to significant reductions in the numbers of laboratory animals used, as well as to the refinement and replacement of laboratory animal procedures, while also producing real benefits for the biomedical sciences.
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News & Views

ATLA Staff Writer

ARDF Campaign to End Use of Ascites Methods
NASA Cancels Monkey Space Research
Call for Proposals for Procter & Gamble Grants
Direct Exposure of Cells In Vitro to Aerosols and Gases
Gillette Grants Awarded
Swiss to Vote on Genetic Engineering
Memorial Service for Clive Hollands
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2017-01-09T06:26:17+00:00 Tags: |

The Development and Validation of Expert Systems for Predicting Toxicity

John C. Dearden, Martin D. Barratt, Romualdo Benigni, Douglas W. Bristol, Robert D. Combes, Mark T.D. Cronin, Philip N. Judson, Martin P. Payne, Ann M. Richard, Milon Tichy, Andrew P. Worth and Jeffrey J. Yourick

This is the report of the twenty-fourth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM’s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals.
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2017-01-09T06:26:17+00:00 Tags: |

The Fourteenth Congress on In Vitro Toxicology of the Scandinavian Society for Cell Toxicology

Björn Ekwall

The Scandinavian Society for Cell Toxicology (SSCT) held its fourteenth annual congress on in vitro toxicology on 5–8 September 1996, in Uppsala, Sweden. The meeting was attended by a total of 52 scientists, of which 33 came from the four Nordic countries, while the other 19 participants represented 11 other countries.
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2017-01-09T06:26:17+00:00 Tags: |

Detoxification Strategies of Two Types of Spiders Revealed by Cypermethrin Application

Søren Achim Nielsen, Jørgen Clausen and Søren Toft

In order to compare the detoxification capacities of two species of spiders, Linyphia triangularis and Pardosa prativaga were exposed to a commonly used pesticide, cypermethrin (a chlorinated pyrethroid). Two detoxification enzyme systems — glutathione Stransferase (GST) and glutathione peroxidase (GSH-Px) — were assayed as biomarkers. The two species of spiders have different life-styles and eat different food, and in our experiment were at different stages of their life-cycle. Therefore, differences in detoxification strategies could be expected. From our results, we suggest two different strategies. L. triangularis had a generally high basal level of GST, which could be further induced by exposure to cypermethrin. The basal activity of GSH-Px was relatively low, and no response to toxic exposure could be traced. Thus, this enzyme plays only a minor role in the detoxification process of cypermethrin in L. triangularis. In P. prativaga, a low basal GST activity was found. However, the basal level of GSH-Px was comparable to that of L. triangularis. A significant rise in GSH-Px activity (with t-butyl-hydroxyperoxide as substrate) was apparent 12 hours after exposure. This may indicate that P450 enzyme systems are involved in the cypermethrin metabolising process in P. prativaga. In addition, GSH-Px activity (with H2O2 as substrate) could be traced in P. prativaga but not in L. triangularis, indicating that a true peroxidase system is active in P. prativaga.
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An Ultrastructural Evaluation of the SKIN2TM ZK1200 System as an In Vitro Model of the Human Oral Mucosa

Dorthe Arenholt-Bindslev, Lis Andersen Torpet and Kaj Josephsen

At present, there are no validated experimental models for the preclinical oral mucosa irritancy screening of dental materials and oral hygiene products. Standardised in vitro models for the assessment of skin irritancy have been marketed, and have made possible the application of such models in preclinical skin irritancy testing. Some of these are currently undergoing international interlaboratory validation. The aim of the present study was to evaluate the histology of the commercially available SKIN2TM ZK1200 system as a potential in vitro model of the human oral mucosa. Histological evaluation by light microscopy and transmission electron microscopy over 6 days revealed a fibroblast-rich matrix covered by an 8–12 cell layer of stratifying epithelium, which shared a number of basic characteristics with non-keratinised human oral mucosa (for example, desmosomes, dispersed tonofilaments, glycogen deposits, membrane-coating granules, and keratohyalin granules, both the spherical and ribosome-associated types). Exposure to a methacrylate-based dental fissure sealant for 24 hours caused structural changes in the epithelium which were not reflected by a cytochemical cytotoxicity assay (MTT).
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Cadmium-induced Inhibition of ADH stimulated Ion Transport in Cultured Kidney derived Epithelial Cells (A6)

Henning F. Bjerregaard and Brian Faurskov

An epithelial cell line (A6) derived from the distal tubule of toad kidney, was used to study the effect of cadmium (Cd2+) on the increase in active ion transport induced by antidiuretic hormone (ADH). Addition of Cd2+ (1mM) to the basolateral solution of A6 epithelia generated an immediate and transient increase in active ion transport, measured as short circuit current (SCC). This increase was not affected by prior addition of ADH. However, there was a distinct inhibition of ADH-induced stimulation of SCC in epithelia pre-treated with Cd2+. Since cAMP serves as an intracellular messenger for ADH by increasing the ion permeability of the apical membrane in A6 epithelial cells, the effects of Cd2+ on enzymes involved in cAMP metabolism were measured. The results showed that Cd2+ markedly inhibits cAMP production by inhibiting adenylate cyclase (which had been stimulated with forskolin, magnesium or a nonhydrolysed GTP-analog), indicating that Cd2+ inhibits the catalytic subunit of adenylate cyclase. Furthermore, degradation of cAMP by phosphodiesterase was not stimulated by Cd2+, also suggesting that the mechanism by which Cd2+ inhibits the ADH-induced ion transport could be through inhibition of adenylate cyclase. Taken together, these results indicate that, in addition to the well-known toxic effect on the proximal tubule, Cd2+ could also have an effect on the distal part of the kidney, where the important hormonal regulation of salt and water homeostasis takes place.
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An In Vitro System for Evaluation of Oxidative Stress and the Effects of Antioxidants

Benedikte Brogaard and Jørgen Clausen

Oxidative stress is defined as an imbalance between prooxidants and antioxidants. There is a need for a simple in vitro method for evaluation of the effects of oxidative stress and the effects of antioxidants. In the present study, we used primary cultures of human lymphocytes exposed to either paraquat (PQ) or mitomycin C, two prooxidants generating two different types of free-radicals formed either by P450-reductase or by DT-diaphorase, respectively. The toxicity was measured by estimation of DT-diaphorase and glutathione peroxidase (GSH-Px) activity, and by estimation of the level of malondialdehyde (MDA) as a function of time and increasing doses of the two prooxidants. The enzyme activities were related to both total DNA content and total protein content of cellular homogenate. All estimations were made by exposing human lymphocytes to increasing concentrations (up to 100μM) of the two prooxidants. However, since cellular death occurred at concentrations above 60μM, only data for exposure to concentrations below 70μM are presented. When the enzyme activities were expressed per cellular unit (i.e. per gram DNA) 30μM mitomycin C induced a 30% increase in DTdiaphorase activity. Similarly, a dose-dependent increase (maximum 100% increase) in DTdiaphorase activity was found after exposure to PQ (up to 60μM). Similar data were obtained when data were related to the total protein. Only a minor increase (11%) in GSH-Px activity was induced by 50μM mitomycin C, whereas 20–70μM PQ induced a 41% increase in GSH-Px activity. Both prooxidants induced more than a doubling in the cellular MDA concentration. These findings demonstrate that both DT-diaphorase and GSH-Px are up-regulated during oxidative stress. However, sensitivity to prooxidant-induced stress seems to depend to some extent on the chemistry of the free-radicals generated. Thus, the single-electron pyridium cations generated by PQ seem to be more toxic than the single-electron semi-quinones generated by mitomycin C. The same cellular system was used to evaluate the effects of antioxidants. Quercetin, a naturally occuring flavone, and selenium (sodium selenite), which is an essential part of glutathione peroxidase, were selected. PQ-induced stress and exposure to 5μg/ml quercetin for 4 hours decreased the MDA level in the medium by 11% and in the cells by 33%. PQ-induced stress and exposure to 160μg/l selenium for 18 hours reduced MDA levels similarly, by 19% in the medium and 14% in the cells. Both antioxidants induced a 50% reduction in GSHPx activity.
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5´-Nucleotidase Activity and Galactolipid Accumulation in Triethyllead-exposed Glial Cell Cultures

Inger K. Grundt, Marvelyn Rise and Harald Nyland

The developing nervous system is vulnerable to heavy metal exposure, which can cause alterations in neuronal and glial cells in the brain. Thus, heavy metals such as mercury and lead reduce myelin galactocerebroside (GalC) synthesis, and increase the ratio of non-hydroxylated fatty acids to hydroxylated fatty acids (GalC-N:GalC-OH) in the GalC molecules in newborn rats. This study investigated the effect of lead on the expression of myelin components by the myelin-forming oligodendroglial cells (OG) in vitro. Primary cultures of mixed glial cells from brains of newborn rats were continuously exposed to triethyllead (TEL; 1nM, 10nM, 50nM and 100nM) for 3 weeks, one week after seeding. The first morphological alteration observed was an increased proliferation of OG in cultures exposed to 10nM TEL. Biochemical analyses showed up-regulation of the enzymes, 2´3´-cyclic nucleotide 3´-phosphodiesterase and 5´- nucleotidase. GalC synthesis was also stimulated, and the ratio GalC-N:GalC-OH was reduced. The results indicate that TEL stimulates the differentiation and maturation of OG in cultures, which suggests that the alterations induced by heavy metals in newborn rats are not due to interference with the OG maturation.
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Mechanism of Tamoxifen’s Retinal Toxicity, Studied in Pig Pigment Epithelial Cell Cultures

Hanna Mäenpää, Tarja Toimela, Pirjo Saransaari, Lotta Salminen
and Hanna Tähti

The anticancer drug tamoxifen is widely used in breast cancer therapy. Tamoxifen has been reported to cause ocular toxicity and impairment of vision in epidemiological studies. To study the possible role of an excitotoxic mechanism in the ocular toxicity of tamoxifen, we investigated the effect of tamoxifen on retinal pigment epithelium (RPE) glutamate uptake in vitro. RPE, a layer of cells between photoreceptors and choroidal capillaries, contributes to the regulation of the concentration of the major excitatory amino acid, glutamate, in the subretinal space. Dysfunction in RPE glutamate uptake can lead to accumulation of extracellular glutamate and can cause various excitotoxic effects in the retina. The study was conducted by using cultured pig RPE cells. Six different tamoxifen citrate concentrations, ranging from 1μM to 100μM, and [3H]-L-glutamate were added to the culture medium. To specify the glutamate uptake, 1mM dinitrophenol was added and a Na+-free culture was used. Due to the anti-oestrogenic character of tamoxifen, the possible effect of β-oestradiol on the glutamate uptake of RPE was also examined. The results show that glutamate uptake by RPE cells was reduced in the presence of tamoxifen, and that the reduction was dose-dependent. These results suggest that tamoxifen exposure could lead to the extracellular accumulation of glutamate. Disturbances in glutamate uptake can cause eye toxicity via an excitotoxic mechanism. The glutamate uptake of RPE cells was reduced under Na+-free conditions and was also reduced in the presence of dinitrophenol.
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