ATLA 27.3, May 1999

//ATLA 27.3, May 1999

New Measures on Animal Experimentation in the UK Will Improve Animal Welfare and Scientific Research

Robert D. Combes

On reading the lead article, The Increasing Burden of Regulation, in the April 1999 issue of RDS News,1 I experienced a mixture of emotional states, ranging from disbelief and annoyance to frustration and sadness. In this article, the Executive Director of the Research Defence Society, Mark Matfield, claims that the new measures introduced by the Labour Government to the existing legislation controlling animal experimentation in the UK will improve animal welfare but hinder scientific research. Dr Matfield asserts that the degree of regulation now facing investigators “is seriously impeding some areas of science in the UK”. He believes that the increased focus on animal welfare is not justified in terms of the likely resulting loss of scientific benefits.
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Cellular and Molecular Targets of Benzo[a]pyrene and Metal Toxicity in Xenopus laevis Embryos and in Hep G2 Cells

Marina Camatini, Patrizia Bonfanti, Anita Colombo, Chiara Urani and Silvia Crippa

This paper describes the use of two in vitro systems (stage 35 Xenopus laevis embryos and the human hepatoblastoma cell line, Hep G2) to study effects of some environmental contaminants (benzo[a]pyrene, copper and zinc), which are representative of chemicals with different cell targets and mechanisms of action. The ability to activate benzo[a]pyrene and to metabolise it with the cytochrome P4501A isozyme were demonstrated in both in vitro systems by assessing the formation of water-soluble and protein-bound benzo[a]pyrene metabolites and by immunochemical analysis. In X. laevis embryos, the formation of DNA adducts demonstrated the ability to produce benzo[a]pyrene reactive metabolites. Moreover, in Hep G2 cells, the cytoskeletal protein, tubulin, and the reduced form of glutathione proved to be the cellular targets of copper and zinc toxicity. In response to metal-induced stress in Hep G2 cells, there was a cytoplasmic reorganisation of heat shock protein, Hsp 70. In conclusion, the in vitro systems provide for a rapid evaluation of heterogeneous compounds such as benzo[a]pyrene and heavy metals that differ in toxic potency and mechanisms of action. They could also be used to study the mechanisms of toxic action and to identify specific cellular and molecular targets.
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EDIT: A New International Multicentre Programme to Develop and Evaluate Batteries of In Vitro Tests for Acute and Chronic Systemic Toxicity

Björn Ekwall, Cecilia Clemedson, Barbro Ekwall, Patrik Ring and Lennart Romert

The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) programme provided a battery of three basal cytotoxicity tests with a good (R2 = 0.77) prediction of human acute lethal blood concentrations. The predictive power of this battery would be considerably improved by the addition of new supplementary in vitro tests. The development of these new tests will be facilitated by a close coupling of test development to evaluation. The Cytotoxicology Laboratory, Uppsala (CTLU), is therefore inviting all interested in vitro toxicologists to take part in the Evaluation-guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). All EDIT activities (subprojects) will be designed on a case-by-case basis, but will follow a common pattern. The CTLU will use the accumulated MEIC/EDIT data, and its experience from the previous MEIC evaluation, to suggest priority areas, i.e. the need for certain in vitro toxicity data/tests as supplements to existing in vitro models/batteries on human systemic toxicity. Detailed research programmes corresponding to these areas will be published on the Internet. The CTLU will also try to raise funds for some projects and will coordinate multilaboratory studies. Interested laboratories developing or already using priority tests are encouraged to join the subprojects and to test specific sets of substances (usually sets of MEIC reference chemicals) in their new assays. The CTLU will provide adequate human reference data and will also evaluate results as single components of complex models, together with the laboratory conducting the test. At present, ten priority areas have been identified: a) repeat dose toxicity in vitro; b) urgent mechanistic information from in vitro studies of protein denaturation, morphology of cell injury, differential toxicity between various rapidly measured endpoints (10–60 minutes) and 24-hour cytotoxicity, toxicity to aerobic cells, and discrimination between rapid and slow cytotoxic mechanisms; c) in vitro tests on vitally important, specific receptor toxicity in humans; d) excitatory cytotoxicity; e) reversibility of cell toxicity; f) in vitro tests on passage across the blood–brain barrier; g) in vitro tests on absorption in the gut; h) protein binding in vitro; i) in vitro tests on distribution volumes (Vd); and j) in vitro tests on biotransformation to more-toxic metabolites (hepatocytes plus target cells). This paper gives a short presentation of the rationale for each subproject and reports on ongoing activities.
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The Effects of Oxidative Stress in In Vitro Cultured Astroglial Cells

Casper Møller Frederiksen and Jørgen Clausen

It has been suggested that glial cells in the central nervous system might function as a buffer and protect neurons and synapses. Associated with such a function, glial cells might be affected in degenerative diseases, for example, Alzheimer’s disease and Parkinson’s disease, due to generation of free-radicals. Free-radicals might be generated during the metabolic
transformation of xenobiotics. The purpose of the present study was to determine whether a xenobiotic (in this case, paraquat), is metabolised in glial cells during the generation of freeradicals. Furthermore, this study determined whether free-radicals can induce DNA fragmentation and whether this fragmentation can be repaired. The data produced indicated that astroglial cells contain P450-reductase which transforms paraquat into a pyridium free-radical. In turn, this causes a dose-dependent DNA fragmentation, as determined by using single-cell gel electrophoresis. The dose-dependent effect was valid up to 80μM paraquat. The oxidative stress induced in the astroglial cells was also associated with a maximum 15% increase in the anti-oxidative enzyme, glutathione peroxidase. After exposure to 40μM paraquat, followed by growth of the cells in a paraquat-free medium, DNA repair was shown to be rather slow, and was only obvious two hours after exposure to paraquat. This might be related the shuttle in which paraquat/P450-reductase is implicated, which causes a protracted generation of free-radicals. The data are discussed in relation to the available literature.
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Predicting the Toxicity of Oil-shale Industry Wastewater by its Phenolic Composition

Anne Kahru, Lee Põllumaa, Rain Reiman and Annely Rätsep

The chemical composition and toxicity of five phenolic wastewater samples collected from the Kohtla-Järve (Estonia) oil-shale industry region were analysed. The total phenolic contents (HPLC data) of these samples ranged from 0.7mg/l to 195mg/l. A total of 11 phenolic compounds were found in the wastewater samples, the most abundant being phenol (up to 84mg/l) and p-cresol (up to 74mg/l). Artificial phenolic mixtures were also composed, to mimic the content of phenolic compounds in the wastewater samples. The theoretical toxicities of these artificial mixtures were calculated by using the toxicities of the individual phenolic constituents to photobacteria (the BioToxTM test) and were assumed to have an additive mode of action. From the BioTox data, the additive toxic effects of phenolic compounds in the artificial mixtures were confirmed to be highly probable. The toxicities of the wastewater samples and their artificial phenolic analogues (mixtures) were studied by using a battery of Toxkit microbiotests (Daphtoxkit FTM magna, Thamnotoxkit FTM, Protoxkit FTM and Rotoxkit FTM) and three photobacterial tests (MicrotoxTM, BioToxTM and Vibrio fischeri 1500). The wastewaters were classified as toxic (two samples), very toxic (two samples) and extremely toxic (one sample). Comparison of the test battery responses showed that the industrial wastewaters were 2–28-fold more toxic than the respective artificial phenolic mixtures. The photobacterial tests proved to be the most appropriate for screening purposes. This was the first attempt to use a test battery approach in the toxicity testing of Estonian wastewaters. The study showed that the toxicity of oil-shale industry wastewaters could not be predicted solely on the basis of their phenolic composition, since only 7–50% of their toxicity was shown to be due to phenolic compounds. It is true, to a certain extent, that the majority of environmental samples are usually very complex and contain various types of pollutants. As even a full chemical analysis (which is very expensive) can easily miss the constituent(s) with the greatest toxic effect(s), the use of toxicity tests in parallel to chemical analysis should be encouraged.
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Can Cytotoxic Effects Induced by Industrial Chemicals be Time-dependent?

Ewa Kuchowicz and Konrad Rydzynski

Short-term and delayed cytotoxic effects of selected water-soluble and waterinsoluble industrial chemicals (dimethyl sulphoxide, ethyl alcohol, methyl alcohol, ammonium nitrate, benzalkonium chloride, butoxyethanol and propylene glycol) were tested on confluent 3T3-L1 mouse fibroblasts by using the neutral red uptake (NRU) assay and the MTT assay. The NRU and MTT assays were performed after exposure to a chemical for 10 minutes and 3 hours and then again 7 days later. The results indicate that the system of testing used permits the assessment of both early and delayed cytotoxic effects of different classes of chemicals. Our experiments revealed that three out of the seven substances tested (butoxyethanol, dimethyl sulphoxide and propylene glycol) exhibited similar cytotoxic effects when assessed after exposure for 10 minutes and 7 days later. The results for ammonium nitrate, ethyl alcohol and methyl alcohol, tested after exposure for 10 minutes and 7 days later, did not significantly differ from each other. However, we noted that the cytotoxic effects observed 7 days after exposure were more pronounced than those found for the same concentration, after exposure for 3 hours. One chemical tested (benzalkonium chloride) had almost no cytotoxic effect after exposure for 10 minutes and 3 hours, but showed a strong cytotoxic effect 7 days after exposure. Our study indicated that the cytotoxic effect might be time-dependent in some chemicals, thus inducing a delayed effect in vitro. In our opinion, it is advisable to assess the cytotoxicity of a chemical after exposure for 3 hours (for immediate effects) and again 7 days later (for delayed effects).
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Phytotoxicities of Selected Chemicals and Industrial Effluents to Nitellopsis obtusa Cells, Assessed by Using a Rapid Electrophysiological Charophyte Test

Levonas Manusadzianas, Rimantas Vitkus, Ralf Pörtner and Herbert Märkl

The acute phytotoxicities of seven heavy metals (Cd2+, Cu2+, Hg2+, Ni2+, Zn2+, Cr6+ and Co2+), three phenolic compounds (phenol, 3,5-dichlorophenol and pentachlorophenol) and nine industrial effluents were appraised by using a rapid electrophysiological test with cells of the charophyte, Nitellopsis obtusa. The EC50 values (concentrations causing a 50% decrease in resting potential) obtained for reference chemicals were compared with those of five microbiotests (Polytox®, Microtox®, Selenastrum capricornutum growth inhibition, Daphnia magna immobilisation and Rotoxkit FTM) taken from the scientific literature. The 45-minute charophyte test, the freshwater Algaltoxkit FTM, Daphtoxkit FTM and Rotoxkit FTM were conducted simultaneously to assess the toxicities of effluents. The Toxkit microbiotests were typically two orders of magnitude more sensitive than the electrophysiological charophyte test to pure chemicals. The electrophysiological charophyte test was generally more sensitive than the Toxkit microbiotests to complex effluents. The rapid electrophysiological test, employing the 45- minute membrane depolarisation of N. obtusa cells as an endpoint, demonstrated similar sensitivity to heavy metals and phenolic compounds as the 20-minute bacterial Polytox® test, but less sensitivity than the 15-minute Microtox® test. Therefore, this rapid macroalgal test appears to be valuable as a sublethal toxicity screening tool for effluents.
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Oxidative DNA Damage in Human Cells Induced by Paraquat

Helena Petrovská and Mária Dušinská

Oxidative DNA damage was studied after exposing two human transformed cell lines (HeLa and Hep G2) and freshly isolated human peripheral lymphocytes to the herbicide, paraquat. We used the alkaline comet assay, modified by incubating nucleoid DNA with endonuclease III to detect oxidised pyrimidines, and with formamidopyrimidine glycosylase to detect 8-oxo-guanine and ring-opened purines. Paraquat induces both strand breaks and oxidised bases, the amounts of each being dependent on the concentration of paraquat and the cell type exposed. Exposure to lower concentrations of paraquat for 1 hour induced dose-dependent DNA damage in Hep G2 cells and in human peripheral lymphocytes. DNA damage was reduced at higher concentrations. Our results support the finding that paraquat induces oxidative stress but, over a certain concentration range, also stimulates antioxidant protection. Reduction of DNA damage was not found in HeLa cells after exposure for 1 hour or 24 hours. Short-term exposure to paraquat induced a moderate amount of oxidative DNA damage (mainly oxidized pyrimidines) in HeLa cells. Exposure for 24 hours induced a high proportion of oxidised bases and strand breaks. Hep G2 cells showed the greatest number of DNA strand breaks, with no sign of base oxidation.
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Toxicity of Mercury to Hybridoma TA7 Cells

Inessa Remez, Pauls Andersons and Hackel Veksler

Environmental mercury and mercury compound contamination has increased dramatically since the industrial revolution. This paper describes the toxic effects of mercury on a culture of hybridoma TA7 cells, which produce antibodies against the A-subunit of viskumin. Cells were cultivated on 96-well flat-bottomed plates with RPMI-1640 medium supplemented with 10% fetal calf serum at 37°C in 5% CO2/95% air. The cells were exposed to 0.1nM/l–10μM/l Hg2(NO3)2.2H2O (mercury nitrate) during the exponential growth phase. Toxicity was assessed by using the colorimetric MTT (tetrazolium) assay after exposure for 48 hours. Cell growth and cell survival were evaluated by using percentage indices of cellular content in exposed cells when compared to non-exposed control cells. The concentrations of the noeffect level, the lowest observed effect level and the the highest toxic effect level were registered. The toxic effects of the mercury compound on the hybridoma cells occurred between 0.1μM/l and 10μM/l.
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