Synthetic organic food colouring agents and their degraded products: effects on human and rat cholinesterases

Most synthetic coloured additives are carcinogenic; teratogenic and cause allergic reactions. In this study, the effects of synthetic azo dyes (sunset yellow FCF and carmoisine), as well as their degraded products (sulphanilic acid and naphthionic acid), on both true and pseudo-cholinesterases (ChEs) are studied. The results indicate that the synthetic azo dyes and their degraded products inhibit both human true and pseudo-ChE activities in vitro. The concentration of coloured additive that cause 50% inhibition (IC50) and enzyme inhibitor dissociation constant (Ki) show that sunset yellow FCF produces greater inhibition of both true and pseudo-ChEs than does carmoisine and sulphanilic acid, while naphthionic acid produces greater inhibition of pseudo-ChE only. Ki indicates that the affinity of sulphanilic acid for both true and pseudo-ChEs is higher than the other three inhibitors. Inhibition of both true and pseudo-ChEs by sunset yellow FCF is of mixed (competitive and non-competitive) type, but carmoisine and sulphanilic acid are non-competitive. Naphthionic acid produces a competitive inhibition kinetic with plasma ChE only. This inhibition is abolished by dialysis, indicating that their effects are reversible. The effects of sunset yellow FCF, carmoisine, sulphanilic acid and naphthionic acid on rat true and pseudo-ChEs are investigated. The data clearly show that there is a significant decrease in enzyme activity. Sulphanilic acid and sunset yellow FCF are the most potent in vivo inhibitors of true ChE and pseudo-ChE, respectively.     

Additives contained in drug formulations most frequently prescribed in Switzerland

The presence of substances known to induce pseudoallergic reactions was investigated by means of a questionnaire to the manufacturers of 1,467 frequently administered formulations. Benzoates were found in 15% of the formulations, sorbates in 5.5%, sulfites in 3.8%, and benzalkonium in 3.0%. The occurrence of the seven artificial colours studied was as follows: indigotin 7.8, erythrosine 7.4, sunset yellow 6.6, tartrazine 4.9, quinoline yellow 2.8%, ponceau (new coccine) 2.6, and amaranth 1.7%. A significant risk of exposure to preservatives and dyes likely to induce asthma, urticaria, or other pseudoallergic reactions exists for all individuals taking commercial drug products.     

Incidence of bronchoconstriction due to aspirin, azo dyes, non-azo dyes, and preservatives in a population of perennial asthmatics.

Forty-five patients with moderately severe perennial bronchial asthma were challenged by ingestion of: acetylsalicyclic acid (ASA); 4 azo dyes (tartrazine, sunset yellow, amaranth, and ponceau); 3 non-azo dyes (erythrosine, brilliant blue, and indigotin); sodium benzoate (NaB); parahydroxybenzoic acid (OHBA); butylated hydroxyanisole (BHA); and butylated hydroxytoluene (BHT). A fall in forced expiratory volume is one second (FEV1) greater than 25% from baseline was considered positive. Seven patients who gave an unequivocal history of aspirin intolerance were not challenged with ASA; an additional 13 had positive open challenges to ASA, giving an apparent incidence of aspirin sensitivity of 20/45. The presence of nasal polyps, simusitis, or the regular use of corticosteroids, either singly or in combination, was not associated with an increased incidence of reactions to ASA. Significant bronchoconstriction to open challenges with agents other than ASA was less frequent. Positive open challenges to all substances except aspirin were followed by double-blind challenges which were positive in only 3 instances: 1 each with erythrosine, ponceau, and NaB/OHBA. Our findings confirm that ASA intolerance is relatively common but suggest on the other hand that reactions to dyes and preservatives are uncommon cause of clinically significant bronchoconstriction in moderately severe perennial asthmatics.     

4-Aryl-4H-chromene-3-carbonitrile derivatives: evaluation of Src kinase inhibitory and anticancer activities

Src kinase mutations and/or overexpression have been implicated in the development of a number of human cancer including colon, breast, and lung cancers. Thus, designing potent and selective Src kinase inhibitors as anticancer agents is a subject of major interest. A series of 4-aryl substituted derivatives of 2-amino-7-dimethylamino-4H-chromene-3-carbonitrile were synthesized using one-pot reaction of appropriate substituted aromatic aldehydes, malononitrile, and 3-(dimethylamino)phenol in the presence of piperidine. All 23 compounds were evaluated for inhibition of Src kinase and cell proliferation in human colon adenocarcinoma (HT-29) and leukemia (CCRF-CEM) cell lines. Among the tested compounds, 2-chlorophenyl- (4c), 3-nitrophenyl- (4h), 4-trifluoromethyphenyl- (4i), and 2,3-dichlorophenyl- (4k) substituted chromenes showed Src kinase inhibitory effect with IC(50) values of 11.1-18.3 μM. Compound 4c was relatively selective against Src (IC(50) = 11.1 μM), when compared with selected kinases, epidermal growth factor receptor (EGFR, IC(50) > 300 μM), C-terminal Src kinase (Csk, IC(50) = 101.7 μM), and lymphocyte-specific protein tyrosine kinase (Lck, IC(50) = 46.8 μM). 3-Chlorophenyl substituted thiazole (4v) and 2-chlorophenylsubstituted thiazole (4u) chromene derivatives inhibited the cell proliferation of HT-29 and CCRF-CEM by 80% and 50% respectively, at a concentration of 50 μM. The data indicate that 4H-chromene-3-carbonitrile scaffold has the potential to be optimized further for designing more potent Src kinase inhibitors and/or anticancer lead compounds.     

Antileishmanial and antimalarial chalcones: synthesis, efficacy and cytotoxicity of pyridinyl and naphthalenyl analogs

The antileishmanial and antimalarial activity of methoxy-substituted chalcones (1,3-diphenyl-2-propen-1-ones) is well established. The few analogs prepared to date where the 3-phenyl group is replaced by either a pyridine or naphthalene suggest these modifications are potency enhancing. To explore this hypothesis, sixteen 3-naphthalenyl-1-phenyl-2-prop-1-enones and ten 1-phenyl-3-pyridinyl-2-prop-1-enones were synthesized and their in vitro efficacies against Leishmania donovani and Plasmodium falciparum determined. One inhibitor with submicromolar efficacy against L. donovani was identified (IC50 = 0.95 microM), along with three other potent compounds (IC50 < 5 microM), all of which were 3-pyridin-2-yl derivatives. No inhibitors with submicromolar efficacy against P. falciparum were identified, though several potent compounds were found (IC50 < 5 microM). The cytotoxicity of the five most active L. donovani inhibitors was assessed. At best the IC50 against a primary kidney cell line was around two-fold higher than against L. donovani. Being more active than pentamidine, the 1-phenyl-3-pyridin-2-yl-2-propen-1-ones have potential for further development against leishmaniasis; however it will be essential in such a program to address not only efficacy but also their potential for toxicity.     

Carmine-picroindigocarmine: an alternative multiple staining method.

Carmine-picroindigocarmine, a multiple staining method, was developed at the beginning of the previous century by Professor Harry Kull at the University of Tartu. The stain, combining copper carmine, picric acid and indigocarmine gives bright and colourful results. Nuclear structures are stained in red, cytoplasm in varying shades from yellow to green, collagen fibres in blue, the matrix of hyaline cartilage in greyish-blue, muscle tissues from brownish-red to brownish-green, erythrocytes in yellow. Squamous epithelia are stained in red with horney layers in dark red, nail plate and hairs are stained in bright yellow. The carmine-picroindigocarmine staining is stable, which allows for the combining of additional dyes without interfering with the main colouring. The combination of carmine-picroindigocarmine staining with resorcin-fuchsine in principle maintained the colouring of Kull's original method with additional staining of elastic fibres in violet colour that clearly differentiated them from blue stained collagen fibres. The described multiple staining gives an original colourful and aesthetic result, providing an alternative to other multiple staining methods.     

Fluorescence-based antiviral assay for the evaluation of compounds against vaccinia virus, varicella zoster virus and human cytomegalovirus

Recombinant vaccinia virus (VACV), varicella zoster virus (VZV) and two human cytomegaloviruses (HCMV) expressing the green fluorescent protein (GFP) and the enhanced yellow fluorescent protein (EYFP) were used to develop a fluorescence-based assay for testing antiviral compounds. Infection of human embryonic lung fibroblasts (HEL) with the different recombinant viruses produced stable and detectable amount of GFP and EYFP signal as quantitated by automated fluorometry. The sensitivity of the recombinant viruses to a panel of antiviral drugs was measured and the fluorescence-based assay was compared to the cytopathic effect reduction assay (CPE-RA) in case of VACV and HCMV or to the plaque reduction assay (PRA) in case of VZV. The 50% inhibitory concentration (IC(50)) values for reference anti-pox and anti-herpesvirus compounds were comparable to those determined by CPE-RA or PRA assays. Furthermore the fluorimetric data could be confirmed by a flow cytometry assay. GFP- and EYFP-recombinant viruses proved to be a convenient tool for the evaluation of antiviral agents.     

Human embryonic cell growth assay for teratogens with or without metabolic activation system using microplate

In vitro microassay for the screening of teratogens was investigated on cancer chemotherapeutic agents sterigmatocystins and benzimidazoles using human embryonic palatal mesenchymal (HEPM) cells. Five thousand cells were inoculated into each well of 96-well microtiter plates, and cultivated for 24 hr, after which the media were changed with new ones that contained various amounts of chemicals; after cultivation for an additional 72 hr, the media were discarded, and cells attached to the tissue plate were fixed and stained with Giemsa's solution; the cell number then was counted by colony counter with three readings for each well. For the metabolic activation, the liver S9 obtained from rats pretreated with phenobarbital and 5,6-benzoflavone and cofactors (S9 mix) were added directly to the HEPM cell cultures along with chemicals. After 6 hr, the cultures were exchanged with a fresh medium and incubated for a further 72 hr. The final IC50 (the concentration that inhibits growth 50%) concentration-finding run had 7 to 11 concentration points (mean, three to four wells). Concentrations of the cancer chemotherapeutic agents that inhibited growth by 50% ranged from 0.001 to 10 micrograms/ml. Sterigmatocystins indicated strong inhibition; among three derivatives, O-acetyl sterigmatocystin was the most potent inhibitor. Benzimidazoles also exhibited an inhibitory action on HEPM cell growth; nitro and chloro groups at the 5 position in 2-(2-pyridyl)benzimidazole were found to be potent substituents. As for the activation of cyclophosphamide in the HEPM cell culture, IC50 was decreased to 1.0 ug/ml by the incubation with S9 mix for 6 hr under our experimental conditions, and sterigmatocystin was found to be activated by S9 mix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and antitumor activity of aromatic camptothecin esters

Twenty-eight new aromatic esters of camptothecins 2-29 were prepared in yields of 5 to 96% by straight acylation of camptothecin (1a) and 9-nitrocamptothecin (1b) with various aromatic acids as acylating agents. All of these esters were tested against 14 different human cancer cell lines. The antitumor activity of these compounds was related to the nature of the substituting groups of their side aromatic chains. In general, esters with strong electron-withdrawing groups on their side aromatic chains were active; esters with halogen-substituted side aromatic chains were slightly active; and esters without any substituting groups on their side aromatic chains were practically inactive. The IC50 studies showed that the majority of these esters were not as potent as their parental compounds 1a and 1b; whereas, the potencies of esters 6 and 25 were exceptionally high, much higher than the commercial camptothecin analogues and comparable to (or slightly more potent than) their parental compounds.     

Impact of impurities on IC50 values of P450 inhibitors

During early drug discovery, the synthetic pathways for test compounds are not well defined and impurities in the test compounds are inevitable. Compounds undergo serial screening tests at this stage to assess their biological activities and drug-like properties. Impurities in the test compounds can produce false positive results and therefore complicate the interpretation of data. P450 inhibition is one of the screens used in the early drug discovery process to assess the potential of drug-drug interactions caused by the inhibition of P450 enzymes. The impact of impurities on P450 inhibition has not been investigated. In this study, the impact of impurities on CYP2D6 IC(50) values was evaluated using model compounds. Cimetidine was chosen as the test compound. Quinidine, fluoxetine, fluvoxamine, and ibuprofen were chosen to represent impurities as they inhibit CYP2D6 to varying degrees. The IC(50) values of these model impurities for CYP2D6 were 0.11 μM, 0.98 μM, 13.4 μM, and >100 μM, respectively. Impurities with potent CYP2D6 inhibition, such as quinidine, can significantly decrease the apparent IC(50) value for the mixture. With the addition of only 2% quinidine to cimetidine (mol/mol), the apparent IC(50) value of cimetidine decreased from 98 μM to 4.4 μM. With the addition of 10% quinidine, the apparent IC(50) decreased to 1.04 μM. Such a significant decrease in apparent IC(50) values can produce a false alert and cause the inappropriate elimination of good compounds at an early stage. Impur6ities with low inhibitory potential, such as fluvoxamine and ibuprofen, did not cause a significant change in apparent IC(50) values. An impurity can have a similar effect on the IC(50) values for inhibition of other biological activities. The effect of an impurity on apparent IC(50) values can be predicted by using a simulation curve if the potency of the impurity is characterized.     

CI-922--a novel, potent antiallergic compound--I. Inhibition of mediator release in vitro

CI-922 (3,7-dimethoxy-4-phenyl-N-1H-tetrazol-5-yl-4H-furo[3,2-b]-indole- 2-carboxamide, L-arginine salt) is a novel antiallergy compound which inhibits the release of the inflammatory mediators histamine and leukotriene (LT) from stimulated cells. CI-922 showed potent, effective inhibition of antigen-induced mediator release from human basophils and isolated guinea pig lung. The drug inhibited ragweed or housedust-induced histamine release from basophils of allergic human donors (IC50 = 8.6 microM). The antiallergy agents proxicromil (IC50 = 80 microM) and cromolyn (100 microM) were less potent than CI-922 or inactive, respectively. In fragmented lung from actively sensitized guinea pigs, CI-922 (IC50 = 1.5 microM), blocked the antigen-induced production of LT and was a more potent inhibitor of histamine release (IC50 = 13.4 microM) than proxicromil (IC50 = 72.9 microM), or cromolyn (inactive at 1 mM). CI-922 (IC50 = 0.9 microM) completely inhibited repeated contractions of guinea pig lung strips that were induced by low antigen concentration in the presence of antihistamine (H1). Nordihydroguaiaretic acid (NDGA) (IC50 = 2.8 microM), proxicromil (IC50 = 6.2 microM) and the LT antagonist FPL-55712 (IC50 = 3.3 microM) also were fully effective, but cromolyn (300 microM) was inactive. In other experiments, CI-922 (IC50 = 7.0 microM) inhibited a strong, nonrepeatable lung contraction induced with high antigen concentration (histamine responses blocked), and was six times more potent than FPL-55712. Other investigations in isolated tissue preparations showed CI-922 to be a weak inhibitor of LT or histamine-induced effects with no anticholinergic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the in vitro antiretroviral potential of some Biginelli-type pyrimidines

Despite the success of highly active antiretroviral therapy, AIDS still remains as one of the most important world health problems. Toxicity of current available drugs and inevitable emergence of multi-drug resistant strains makes things worse. In the present study a series of novel Biginelli-type pyrimidine compounds were evaluated as potential anti-human immunodeficiency virus (HIV)-1 agents using green fluorescence protein (GFP) reporter single round HIV-1 infection assay. The rate of infected cells was monitored by flowcytometry. The effect of compounds on the cellular proliferation was considered as the cyotoxicity. The anti-HIV-1 active compounds were selected for HIV-1 replication and syncytium formation assays. The antiretroviral activity of compounds was measured against luciferase reporter A murine leukemia virus (AMLV) virions as the retrovirus control. Compounds 2, 5, 6, 8, 11, 12, 13, 17, 18, 20, and 21 were the most potent against HIV-1. Compound 8 had the 50% inhibitory concentration (IC50) of 100 nmol/l for inhibiting HIV-1 replication and 50% cytotoxic concentration (CC50) was up to 100 μmol/l (therapeutic index (TI) >1000). Results show that the active compounds were able to inhibit the retrovirus control as well. Analysis of structure of the studied compounds proved relationships with their anti-HIV-1 effects. Some of the studied compounds seem to be promising anti-HIV-1 drug candidates. Structural manipulation based on the well-defined structure-activity relationships might propose some new leads for anti-HIV-1 drug discovery programs.     

Synthesis and biological evaluation of 2-, 3-, and 4-acylaminocinnamyl-N-hydroxyamides as novel synthetic HDAC inhibitors

A new series of 2-, 3-, and 4-acylaminocinnamyl-N-hydroxyamides 1-3 have been prepared, and their anti-HDAC (against maize HD2, HD1-B, and HD1-A enzymes) activities have been assessed. Cinnamyl-hydroxyamides bearing acylamino substituents at the C2 position of the benzene ring (compounds 1a-g) showed very low HDAC inhibiting activities, with IC(50) values in the high micromolar range. By shifting the same acylamino groups from C2 to C3 (compounds 2a-g) as well as C4 (compounds 3a-f) position of the benzene ring, a number of highly potent HDAC inhibitors have been obtained. In the anti-HD2 assay 3c (IC(50) = 11 nM) was the most potent compound, being >11600-, 4.5-, and 10-fold more potent than sodium valproate, SAHA, and HC-toxin, respectively, and showing the same activity as trapoxin. HD1-B and HD1-A assays have been performed to screen the inhibitory action of 1-3 against mammalian class I (HD1-B) and class II (HD1-A) HDAC homologous enzymes. From the corresponding IC(50) data, a selectivity ratio has been calculated. In general, compounds 1-3 showed no or little selectivity towards the class II homologue HD1-A, the most selective being 2a with class II selectivity ratio = 4.3. About the inhibitory potency, the 4-(2-naphthoylamino)cinnamyl-N-hydroxyamide 3f showed the highest inhibiting effect against the two enzymes (IC(50-HD1-B) = 36 nM; IC(50-HD1-A) = 42 nM). Selected 2 and 3 compounds will be evaluated to determine their antiproliferative and cyto differentiating activities on HL-60 cells.     

Effect of adenosine analogues and cAMP-raising agents on TNF-, GM-CSF-, and chemotactic peptide-induced degranulation in single adherent neutrophils.

Adenosine and adenosine analogues are potent inhibitors of the respiratory burst in neutrophils. Most investigators, however, have found little or no effect of these compounds on neutrophil degranulation from cytochalasin B-treated neutrophils in suspension. We have instead investigated the effect of adenosine and 2-chloroadenosine on degranulation in adherent neutrophils in the absence of cytochalasin B. Both adenosine and 2-chloroadenosine were effective inhibitors of lactoferrin secretion induced by the chemotactic peptide N-formyl-methionine-leucyl-phenylalanine (fMLP) [50% inhibitory concentration (IC50) of less than 10(-6) M]. Secretion induced by tumor necrosis factor (TNF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was inhibited only at high concentrations (IC50 of approximately 10(-4) M). In the presence of cytochalasin B no inhibitory effect of 2-chloroadenosine was seen. The effect of cAMP-raising agents on secretion from adherent neutrophils was also investigated. Dibutyryl cAMP at 0.2 mM reduced secretion in response to fMLP by 50% but did not inhibit TNF- and GM-CSF-induced degranulation. At a concentration of 2.0 mM dibutyryl cAMP also inhibited exocytosis in response to the two cytokines. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 300 microM reduced fMLP-induced degranulation, whereas a concentration of 1 mM was required to inhibit TNF- and GM-CSF-mediated secretion. The adenylate cyclase activator forskolin (50 microM) alone did not inhibit secretion in response to TNF or fMLP. However, in combination with IBMX (300 microM), forskolin (50 microM) reduced both TNF- and fMLP-induced secretion to less than 10%. PMA-induced exocytosis was unaffected by all these agents. In conclusion, adenosine appears to be an effective inhibitor of neutrophil granule protein secretion induced by fMLP but only a weak inhibitor of exocytosis in response to TNF or GM-CSF. Secretion in response to fMLP was also found to be more susceptible to a rise in cAMP than degranulation induced by TNF and GM-CSF.     

The effect of Ca2+ on the pharmacological specificity of [3H]glutamate binding sites in rat hippocampus

The inhibition of [3H]glutamate binding to rat hippocampal membranes by various compounds was measured. L-Glutamic, L-2-amino-4-phosphonobutyric and 4-fluoroglutamic acids had similar IC50 values (2.12 microM, 4.98 microM and 3.90 microM respectively). D-Glutamate and D-2-amino-4-phosphonobutyrate were less potent than L-glutamate (IC50S 18.9 microM and 46.5 microM respectively) and L-glutamate diethylester (IC50 519 microM) was the weakest inhibitor tested. Addition of 10 mM CaCl2 caused a 2.27-fold increase in displaceable binding but did not alter the IC50 values of any of the inhibitors tested.     

The naphthalenesulphonamide calmodulin antagonist W7 and its 5-iodo-1-C8 analogue inhibit potassium and calcium currents in NG108-15 neuroblastoma x glioma cells in a manner possibly unrelated to their antagonism of calmodulin.

Patch clamp techniques were used to record voltage-sensitive calcium and potassium currents from NG108-15 cells. N-(6-aminohexyl)-5-chloro-1-naphthalene- sulphonamide (W7), a calmodulin (CaM) antagonist and its more potent (10 times) 5-iodo-1-C8 analogue (J8) inhibited these currents in a dose-dependent manner. The inhibition was not dependent on internal or external Ca2+. W7 was about four times more potent as an inhibitor of the transient potassium current (IC50 = 8 microM) than of the M-current or of the calcium current. J8 was also selective for the potassium currents (IC50 values: transient current 4 microM, M-current 11 microM) compared to the calcium current (IC50 36 microM). It is suggested that the inhibition does not result from an anti-CaM action of the compounds.     

Mutagenic screening of marker grenade dyes by the Salmonella reversion assay, L5178Y/TK+/- mouse lymphoma assay, and in vivo sister chromatid exchange analysis in mice

Two dyes (C.I. Solvent Yellow No. 33 and a mixture of C.I. Solvent Yellow No. 33 and C.I. Solvent Green No. 3) were tested for mutagenicity in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay, and also for sister chromatid exchange (SCE) induction in vivo in C57B1/6J mice. In addition, a greater than 99.9% pure sample of the yellow dye [2-(2'-quinolyl)-1,3-indandione] was tested with and without exogenous activation in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay. Neither C.I. Solvent Yellow No. 33 nor the C.I. Solvent Yellow No. 33 and Solvent Green No. 3 mixture was positive for inducing SCEs in vivo. All three dyes were tested in the standard plate incorporation test in seven Salmonella strains TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. The dyes were negative with and without exogenous activation in TA98, TA1535, and TA1538. One test with TA1537 was positive with the greater than 99.9% purified yellow dye. All three dyes gave weakly positive results (less than a twofold increase) with S-9 in TA100 and were clearly positive in TA102 and TA104 both with and without S-9. They also induced mutation at the thymidine kinase locus in mouse lymphoma cells, produced both large- and small-colony trifluorothymidine-resistant mutants, and were clastogenic. The purified yellow dye was further tested for SCE induction in mouse lymphoma cells and was determined to give a slightly positive response in the presence of S-9.     

Design, synthesis, and preliminary evaluation of new pyrrolidine derivatives as neuraminidase inhibitors

A series of pyrrolidine derivatives were designed and synthesized in good yields starting from commercially available 4-hydroxy-L-proline using a suitable synthetic strategy. And their ability to inhibit neuraminidase was evaluated. These compounds showed potent inhibitory activity against influenza A (H3N2) neuraminidase. Within this series, four compounds, 6e, 9c, 9f and 10e, have the good potency (IC(50)=1.56 approximately 2.40microM) which is compared to the NA inhibitor oseltamivir (IC(50)=1.06microM), and could be used as lead compound in the future.     

Cytotoxicity assessment of three therapeutic agents,cyclosporin-A,cisplatin and doxorubicin,with the ciliated protozoan Tetrahymena pyriformis

Cyclosporin-A, a drug possessing potent immunosuppressive properties, is used to prevent allograft rejection. Cisplatin and doxorubicin are two of the pharmaceutical drugs most widely used in cancer chemotherapy. In this study, the cytotoxicological impact of these three therapeutic agents was determined using bioassays performed with a unicellular eukaryote, the ciliated protozoan Tetrahymena pyriformis. For this purpose we used the population growth impairment test and the non-specific esterase activities test. We also examined some morphological effects. The results show that these three agents are toxic towards T. pyriformis. A concentration-dependent inhibitory effect on the cell proliferation rate of T. pyriformis populations was found for the three drugs. The IC(50) values were, respectively, 42.03+/-4.64, 124.37+/-7.47 and 74.62+/-6.12 microM for cyclosporin-A, cisplatin and doxorubicin. Non-specific esterase activities were also modified compared with untreated cells. The IC(50) values were, respectively, 88.32+/-8.35 and 44.61+/-3.33 microM for cisplatin and doxorubicin. Exposure of T. pyriformis to these drugs caused the prompt appearance of digestive vacuoles concentrating particulate elements. This phenomenon was more pronounced at higher concentrations. We also observed deformed cells with cisplatin. T. pyriformis bioassays can offer an alternative in vitro method to cell cultures for the risk assessment of potentially toxic drugs.