Optimisation of the Bovine Whole In Vitro Embryo System as a Sentinel for Toxicity Screening: A Cadmium Challenge

Ellen P.A. Jorssen, Lucia Vergauwen, Karen Goossens, An Hagenaars, Mario Van Poucke, Evi Petro, Luc Peelman, Dries Knapen, Jo L.M.R. Leroy and Peter E.J. Bols

Developmental toxicity testing could greatly benefit from the availability of an in vitro alternative model based on the use of animal embryos that have better human-like physiology than the currently-used alternative models. These current models are insufficient, as extrapolation of the results can be challenging. Therefore, an in vitro bovine embryo culture system was used to expose individual morulae to test substances, and to study developmental characteristics up to the blastocyst stage. Cadmium was chosen as the reference toxicant to investigate the sensitivity of the bovine morulae to various concentrations and exposure times. Oocytes from slaughterhouse-obtained bovine ovaries, were maturated, fertilised and cultured up until the morula stage. Morulae were exposed to different cadmium concentrations for 18 or 70 hours, and developmental competence, embryo quality and the expression of cadmium exposure related genes were evaluated. Cadmium exposure hampered embryonic developmental competence and quality. Compared with the 18-hour exposure, the 70-hour exposure induced a 20-fold higher toxic response with regard to developmental competence and a more ‘cadmium-typical’ transcript expression. The bovine morula might be a promising tool for toxicity testing as, following exposure, the embryos reacted in a sensitive and ‘cadmium-typical’ manner to our reference toxicant.
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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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Increased Heat Shock Protein 70 Expression Following Toxicant-mediated Cytotoxicity: A Ubiquitous Marker of Toxicant Exposure?

Parivash Farzaneh, Abdolamir Allameh, Steven Pratt, Nicholas Moore, Lucy Travis, Elke Gottschalg, Clive Kind and Jeffrey Fry

The up-regulation of heat shock protein (HSP) expression has been proposed as a general biomarker of cellular protection against various environmental stresses and chemicals. The present study investigated the possibility of using HSP70 up-regulation as a biomarker of toxicant exposure in vitro. Cells of a rat hepatoma cell line (FGC4) were exposed to concentrations of 1,3-dichloroacetone, duroquinone, diquat dibromide, menadione, hydrogen peroxide, cadmium chloride (CdCl2) and sodium (meta)arsenite (NaAsO2) that elicited 20–50% cytotoxicity over a 24-hour period, and HSP70 levels were measured by ELISA. Up-regulation of HSP70 expression was demonstrated following treatment with menadione, CdCl2 and NaAsO2, but not with the other chemicals tested. A shorter exposure time (6 hours) and/or the use of non-toxic concentrations reduced the level of HSP70 up-regulation with menadione, CdCl2 and NaAsO2, but did not uncover any up-regulation with the other chemicals. Although the toxicity of the majority of the chemicals tested is believed to involve an oxidative stress component, the results of this study clearly demonstrate that up-regulation of HSP70 expression cannot be used as a general biomarker of toxicant exposure in vitro.
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The Effects of Heavy Metals on Common Carp White Blood Cells In Vitro

Malgorzata Witeska and Marta Wakulska

The in vitro effects of cadmium, copper, lead and zinc, and various cadmium compounds (chloride, sulphate and nitrate) on common carp (Cyprinus carpio) lymphocyte viability and phagocyte activity, were evaluated. The percentage of dead lymphocytes was determined after Trypan blue staining, and phagocyte activity was measured by using the nitroblue tetrazolium (NBT) reduction test. Lead was the most toxic to lymphocytes — the maximum mortality exceeded 30%, and was significantly higher at 1μM of lead, compared to the control. The maximum mortality caused by cadmium was below 10%, but was significantly elevated with 5μM or more of cadmium. Zinc induced lymphocyte mortality from 10μM, whilst no effect was observed with copper. The incubation of full blood with the three cadmium compounds (at 5mg/l of cadmium) showed that cadmium nitrate and cadmium sulphate were more toxic (over 35% and 25% mortality, respectively) than cadmium chloride (about 15% mortality). This was confirmed by the results of tests on isolated cells —1mg/l of cadmium as nitrate and sulphate increased lymphocyte mortality compared to the control and cadmium chloride. Phagocytic activity was less sensitive to heavy metals than was lymphocyte viability. It was significantly reduced following exposure to 50μM and 100μM cadmium, and 100μM zinc, but no effects were observed with either lead or copper.
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Effects of Cadmium on Differentiation and Cell Cycle Progression in Cultured Xenopus Kidney Distal Epithelial (A6) Cells

Henning F. Bjerregaard

Cadmium (Cd) is an important industrial and environmental pollutant, and the kidney is the primary organ to be affected. To elucidate the effects of Cd on cell proliferation, an epithelial cell line (A6) originally derived from the distal part of the Xenopus laevis kidney was cultured in media containing 10% fetal bovine serum. The effects of Cd (added as CdCl2) on cellular growth and differentiation from single cells to confluent epithelia were investigated by visual inspection and by measurement of the degree to which living cells covered a unit area. Over a concentration range from 5 to 50μM, Cd did not affect the settling and adherence of single cells to the bottom of the culture well. The addition of 5μM Cd for 4 days did not affect the ability of the A6 cells to develop confluent epithelia, measured as the area covered by adherent living epithelial cells (99 ± 4% of the control value). However, 10μM Cd did effectively inhibit development of confluent epithelia to 13 ± 5% compared to control. Visual inspection of adherent cells exposed to 50μM Cd for 7 days revealed no increase in cell number or in cell death, which indicated the induction of cell cycle arrest. Flow cytometric analysis showed that treatment of cells with Cd (0.4mM) for 24 hours induced a significant increase in the proportion of G1 phase cells from 58.6 ± 3.9 to 80.6 ± 3.7%, and a corresponding reduction in the proportion of cells in both the S and G2 phases from 24.0 ± 3.6 to 13.4 ± 3.3% and 17.2 ± 1.7 to 5.8 ± 2.1%, respectively. This study showed that Cd stopped cell proliferation in a very narrow concentration range, between 5 and 10μM, and cell cycle analysis indicated that Cd arrested the cells in the G1 phase of the cell cycle.
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