A new low-cost microbiotest with the Protozoan spirostomum teres: culture conditions and assessment of sensitivity of the ciliate to 14 pure chemicals.

This paper defines the culture conditions of the ciliate Spirostomum teres and assesses its sensitivity to some xenobiotics for the development of a new low-cost microbiotest. The model was selected for its ubiquitous distribution, large size for a unicellular species, easy culture in holoxenic medium, moderate generation time, and high sensitivity to pure toxicants. The influence of different culture waters, inocula of ciliates, food, temperature, light, and darkness on the growth of the ciliate population was tested. The shortest generation time (average 39 h) was obtained for cultures incubated at 25 degreesC in the dark with an inoculum of 4 ciliates per ml in 25 ml of Volvic mineral water containing 8 boiled wheat grains, when preincubated without ciliates for the previous week. Under these conditions, it was possible to obtain about 3000 ciliates/ml 3 weeks later. Acute toxicity tests (24-h LC50) were carried out for CuSO4, HgCl2, CdCl2, K2Cr2O7, ZnSO4, Pb(NO3)2, thiram, carbaryl, lindane, parathion, parathion methyl, paraoxon, 2, 4,6-trichlorophenol, and sodium pentachlorophenolate (Na-PCP). Very high sensitivity of the model to Hg2+, Cu2+, Cd2+, thiram, and Na-PCP was established. Comparison of its sensitivity with that of Microtox (current results), Daphnia Magna, Tetrahymena pyriformis, Colpidium campylum, and murine fibroblasts (data from literature) confirms the high sensitivity of the model, especially to heavy metals. Easy-to-perform, cost-effective, and sensitive bioassays using S. teres are suitable for risk assessment and early detection of toxicity in fresh water.

Toxicity assessment of 16 inorganic environmental pollutants by six bioassays.

The relative toxicity of 16 environmental pollutants, such as inorganic elements (Ba, Cd, Co, Cr, Cu, Fe, Ge, Hg, Mn, Nb, Pb, Sb, Sn, Ti, V, and Zn), is evaluated on the L-929 established cell line of murine fibroblasts, with five bioassays [RNA synthesis rate assay (RNA), MTT reduction assay (MTT), neutral red incorporation assay (NRI), Coomassie blue assay, and cellular growth rate assay], and on the ciliated protozoa Tetrahymena pyriformis GL [doubling time of T. pyriformis GL population assay (DTP)]. For each inorganic substance, the six bioassays allowed the toxicological index IC50 ("inhibitory concentration 50%") to be calculated. The IC50 values are useful to rank the tested elements and to compare the features of the six bioassays. The most sensitive assays were the RNA, MTT, NRI, and DTP assays. Moreover, the in vitro IC50 values correlated with the in vivo LD50 values; these results were close to those obtained with established lines of human, murine, or fish cells. The sensitivity and the complementarity of these bioassays would be in favor of their incorporation in a "battery" of tests used for toxicological screening studies of xenobiotics.

Uptake and efflux of polycyclic aromatic hydrocarbons by Tetrahymena pyriformis: evidence for a resistance mechanism.

The action of benzo(a)pyrene (BP), 3-methylcholanthrene (3MC), benzanthracene (BA), and 7,12-dimethylbenzanthracene (DMBA), four polycyclic aromatic hydrocarbons (PAHs), was studied on the unicellular protozoan Tetrahymena pyriformis. This ciliate was exposed to the PAHs at 1, 15, and 37 microM for up to 6 h. BP and BA caused a slight inhibition of cell growth, whereas 3MC and DMBA showed no detectable effect. Cell viability remained unaffected by the PAHs at all concentrations and exposure times tested. Cellular accumulation of PAHs was studied using flow cytometry. The results show immediate accumulation followed by rapid elimination of the compounds. BP uptake was also studied in the presence of verapamil and cyclosporin, compounds known as inhibitors of the multidrug resistance (MDR) pump. In the presence of verapamil, BP was accumulated in larger amounts in cells. With cyclosporin, the accumulation of the PAH was several times higher than under control conditions. The results of GC/MS analysis show that PAH elimination was not linked to biotransformation. These results suggest that the resistance of Tetrahymena against PAH cytotoxicity may be attributed to the rapid efflux of these agents from the cells via an efflux pump probably of the MDR type.

Epinigericin toxicity towards Tetrahymena pyriformis GL; changes in cell volume and intracellular pH.

A study of the toxicity of epinigericin, an antibiotic ionophor, towards the ciliate Tetrahymena pyriformis showed that this molecule stopped cell division, increased cell volume and led to a more basic intracellular pH. The action of epinigericin was probably linked to its function as an ionophor. The ionic selectivity of this molecule is still not known. The raising of the intracellular pH of ciliates by this antibiotic may be linked to its toxic action and its iontransport mechanism in Tetrahymena.

Microplate technique for screening and assessing cytotoxicity of xenobiotics with Tetrahymena pyriformis.

The microplate technique (MT) is developed and compared to the usual flask technique (FT) of Tetrahymena pyriformis GL cultures for assessing toxic effects of organic and inorganic substances on this ciliated protozoa model. Applied to the IC50 determination of 29 xenobiotics, the MT demonstrated good correlation with the FT (Y = 0.991X + 0.012, r = 0.94, P < 0.0001). The MT is rapid, easy-to-handle, inexpensive, and statistically reliable and, so, may be helpful for screening investigations of water-soluble xenobiotics.

Comparative study of two in vitro models (L-929 fibroblasts and Tetrahymena pyriformis GL) for the cytotoxicological evaluation of packaged water.

The preservation of 234 samples of water packaged in polychloride vinyl (PVC), polyethylene terephthalate (PET) and glass-bottles was concurrently evaluated using both classical chemical analysis oriented to finding organic and inorganic substances which could have been leached by the packaging and in vitro toxicological bioassays using two models: the L-929 established cell line of fibroblasts and the ciliated protozoa Tetrahymena pyriformis GL. Although no significant abnormality was detected with the chemical analysis, cytotoxicity was detected with both models in some samples of water bottled for > 18 months, for either PVC, PET or glass-packaging. However, statistical analysis did not allow a mathematical relationship between the cytotoxic effects, the length of storage and the packaging to be demonstrated.

Change in intracellular pH in Tetrahymena pyriformis GL in relation to the toxicity level of the lonophorous antibiotic nigericin.

Nigericin, a carboxylic poly ether antibiotic, is toxic towards Tetrahymena pyriformis GL [1, 7, 12, 13]. Nigericin transports K(+) across biological membranes along the gradient. This transport is generally equilibrated by an influx of H(+) ions. The toxicity of nigericin (at low 10 mg · l(-1) and high 30 mg · l(-1) levels) was studied by measuring the intracellular pH of Tetrahymena pyriformis GL by means of two labelled molecules: (14)C-DMO and (3)H-inulin. This measurement of pHi requires prior knowledge of the number of cells and their volume. The results reveal a correlation of the toxicity of nigericin, acidification of the intracellular environment and increase in cell volume leading to the return of pHi to control values.

Microbial conversion of lonophorous antibiotic nigericin by the ciliate Tetrahymena pyriformis.

The ionophorous antibiotic nigericin is toxic towards the ciliate Tetrahymena pyriformis GL. At the high concentration of 30 mg/l (sublethal dose), it is bioconverted into several products. The yield of one of these (N1) is appreciably improved with pretreated cells, higher concentrations of nigericin (50 or 100 mg/1), and the presence of a larger ciliate biomass. A structural study of N1 by FAB mass spectrometry combined with tandem mass spectrometry and nuclear magnetic resonance shows it to be an epimer, epinigericin, about half as toxic as nigericin. Bioconversion of nigericin by T. pyriformis may be regarded as a detoxication process.

The major pterin in Tetrahymena pyriformis is 6-(D-threo-1,2,3-trihydroxypropyl)-pterin (D-monapterin) and not 6-(L-threo-1,2-dihydroxypropyl)-pterin (ciliapterin).

The major pterin in Tetrahymena pyriformis, strain W, earlier suggested to be L-threo-biopterin and named ciliapterin [1] is now identified as D-threo-neopterin (D-monapterin). This is the first example of a natural D-monapterin. This compound was characterized by its chromatographic behavior, its fluorescence properties and by its oxidation product with alkaline permanganate. The final identification was obtained by comparison with an authentic material using an exchange ligand chromatography method with D-phenylalanine as chiral modifier and Cu (II) as metal ion. D-monapterin is also present as the major pterin in Tetrahymena pyriformis strains GL and ST, and in Tetrahymena thermophila.

Rapid quantification of planktonic ciliates: comparison of improved live counting with other methods.

THE FOLLOWING EFFICIENT AND QUANTITATIVELY VALID METHOD TO FILTER CONCENTRATE AND COUNT LIVE PLANKTONIC CILIATES WAS DEVELOPED AND COMPARED WITH OTHER TREATMENTS: unconcentrated (raw) samples and centrifuged samples were counted live, and the effects of five different fixatives (HgCl(2), Lugol's iodine, formaldehyde, glutaraldehyde, and Champy-DaFano) on the counts were monitored. Samples originated from a eutrophic mountain lake (Lake Aydat, near Clermont-Ferrand, France). Overall, live filtered counts were similar to counts of raw samples, but they were significantly higher (2 to 2.3 fold, P < 0.05) by analysis of variance than counts from centrifuged samples. Nevertheless, some taxa, i.e., Halteria and Loxodes spp., were sensitive to filtration. The live filtered counts were also comparable to counts of raw HgCl(2)-fixed and settled samples. HgCl(2) and Lugol fixation consistently gave the highest total counts, while significantly lower counts were always obtained with Champy-DaFano-fixed samples. Losses due to fixation were insignificant for raw samples but were substantial and statistically significant in concentrated samples (15% after filtration and 71% after centrifugation, compared with counts from the corresponding live samples). Live counting of passively filter-concentrated ciliates has many advantages over other methods. It is two to four times quicker and more efficient. Ciliates are recognized with certainty, more species are identified, and enumeration of dead organisms (e.g., tintinnid loricas) is avoided. It should be recommended as a quantitatively valid alternative to classical methods for assessing planktonic ciliate populations.

[Not Available]. / Application de la colonisation d'un substrat artificiel par les Ciliés à l'Étude de la qualité des eaux d'une rivière.

During four different time periods artificial substrates (polyuréthane blocs) were used to sample ciliates in a river receiving different kinds of pollution. 46 species of ciliates were found, belonging to 40 genera. Their distribution and abundance varied greatly both along the river and with time. The saprobic index method was used to localise sources of organic inputs and follow their change with time. Community structures were analysed through specific diversities and rank-frequency diagrams. This allowed the discovery of toxic chemical inputs and their relative intensities. A parallel study of chemical and physical-chemical pollution indicators of the water confirmed the biological results.

[Not Available]. / Action comparée de deux antibiotiques lonophores (Nigéricine et Epinigéricine) sur les teneurs en ADN macronucléaire du cilié Tetrahymena pyriformis GL, en culture synchrone.

Antibiotics (30 mg/1) added at T 60 mn. lead, in a part of the population, to an increase of the macronuclear DNA content resulting from DNA activity during a phase S 2 which begins after the phase G 2. The largest cells have the highest DNA content and the longest generation time. The results are confirmed by autoradiography with tritiated thymidine. The percentage of macronuclear labelling increases from T 150 to a maximum at T 180, and it is greater with nigericin (46%) than with epinigericin (37%). Thus, epinigericin is less toxic than nigericin.

Evidence for a Ca2+-binding protein associated to non-actin microfilamentous systems in two ciliated protozoans.

Electrophoretic distributions of proteins of isolated cortical cytoskeletons from two ciliated protozoans (Isotricha, Polyplastron) were compared in order to reveal any components common to non-actin microfilamentous structures. A low molecular weight protein (Mr approximately 22 kD) characterized by Ca2+-induced shifts in mobility on SDS-polyacrylamide gels was identified in both ciliates. Two-dimensional electrophoretic coordinates and peptide maps of Ca2+-binding proteins from Isotricha and Polyplastron are fairly similar, suggesting conservation of the same molecular species. In addition, an antiserum raised against two proteins (22-23 KD) from the filamentous ecto-endoplasmic boundary of Isotricha, one of which corresponds to the Ca2+-binding protein, cross-reacts specifically with that of Polyplastron. Using an immunogold staining procedure, the Ca2+-binding protein of Polyplastron was shown also to be located in a cortical microfilamentous layer. This protein is probably different from calmodulin. We postulate that it is involved in the control of the ordering of non-actin microfilaments within the cortex of ciliates.

Biochemical and immunological characterization of the microfibrillar ecto-endoplasmic boundary in the ciliate Isotricha prostoma.

The cytoplasm of the ciliated protozoan Isotricha prostoma is compartmented by a continuous fibrillar system made up of a double layer of 4 nm-diameter filaments: the microfibrillar ecto-endoplasmic boundary (EEB). Isolation of this structure after treatment of the cells in a buffer of low ionic strength in the presence of the detergent Triton X-100 evidenced connections linking the two filamentous layers. One dimensional electrophoresis on SDS-polyacrylamide gel of EEB fractions revealed several major proteins with apparent molecular weights between 11 and 23 K. Of these, two neighboring bands of MW22 and 23 K were removed from gels and used as antigens to obtain rabbit antibodies. The antiserum obtained reacted specifically with injected proteins as shown by the technique of immunological detection on nitrocellulose sheets using the peroxidase reaction product. Electron microscopy localization of the antigens with anti-IgG coupled with colloidal gold showed significant labeling of the EEB within the cortex of Isotricha permeabilized with Triton X-100. We hope that the 22-23 K antiserum will prove to be a useful tool for the comparative study of other non-actin filament systems in Protozoa.