"MAY I BORROW YOUR FILTER?"EXCHANGING FILTERS TO COMBAT SPAMIN A COMMUNITY Anurag Garg, Roberto Battiti, Roberto Cascella Technical Report # DIT-05-089 "May I borrow Your Filter?" Exchanging Filters to Combat Spam in a Anurag Garg Roberto Battiti Roberto G. Cascella Dipartimento di Informatica e Telecomunicazioni, Universit a di Trento, Via Sommarive 14, 38050 Povo (TN), Italy.
Effects of the aquatic contaminant human pharmaceuticals and their mixtures on the proliferation and migratory responses of the bioindicator freshwater ciliate tetrahymena
Contents lists available at Effects of the aquatic contaminant human pharmaceuticals and their mixtureson the proliferation and migratory responses of the bioindicatorfreshwater ciliate Tetrahymena Júlia Láng, László K} Department of Genetics, Cell- and Immunobiology, Semmelweis University, PO Box 370, H-1445 Budapest, Hungary " Acute toxic effects of NSAIDs, b-blockers are unlikely to T. pyriformis.
" Antibiotics and Na-diatrizoate do not inhibit Tetrahymena proliferation.
" Chemotaxis is a more sensitive cell physiological response than proliferation.
" In binary pharmaceutical mixtures, the interaction types are concentration dependent.
An increasing attention is paid to the potential harmful effects of aquatic contaminant pharmaceuticals Received 2 December 2011 exerted on both biosystems and humans. In the present work the effects of 14 pharmaceuticals including Received in revised form 23 April 2012 NSAIDs, antibiotics, b-blockers and a frequently used X-ray contrast media on the proliferation and Accepted 16 May 2012 migratory behavior of the freshwater ciliate Tetrahymena pyriformis was investigated. Moreover, the Available online 12 June 2012 mixture toxicity of four selected pharmaceuticals (diclofenac, ibuprofen, metoprolol and propranolol)was evaluated in binary mixtures using full factorial experimental design. Our results showed that the sensitivity of Tetrahymena to NSAIDs and b-blockers (EC 50 ranged from 4.8 mg L1 to 308.1 mg L1) was comparable to that of algal or Daphnia bioassays. Based on these elevated EC 50 values acute toxic effects of these pharmaceuticals to T. pyriformis are unlikely. Antibiotics and the contrast agent sodium-diatrizoate had no proliferation inhibiting effect. Chemotactic response of Tetrahymena was more sensible than proliferation as signiﬁcant chemorepellent action was observed in the environmen- tally realistic concentration range for acetylsalicylic acid, diclofenac, fenoprofen, paracetamol, metopro-lol, propranolol, timolol and trimethoprim (Chemotaxis Index ranged from 63% to 88%).
Mixture toxicity experiments resulted in a complex, concentration dependent interaction type pattern with antagonism being the predominant interaction type (59%) followed by additivity (37%) and syner-gism (4%). Hence the concept of concentration addition validated for NSAIDs in other organisms cannotbe adopted for this ciliate.
In summary authors suggest Tetrahymena as a sensible model of testing aquatic contaminants as well as underline the signiﬁcance using more speciﬁc endpoints to understand the complex mechanismsinvestigated.
Ó 2012 Elsevier Ltd. All rights reserved.
mainly enter the aquatic environment by municipal wastewaterwhere they are disposed or excreted to following metabolization.
The exponentially increasing number of scientiﬁc publications Some of these compounds resist to conventional wastewater treat- dealing with the occurrence of human and veterinary pharmaceu- ment technologies or they may be transformed into toxic products ticals in the aquatic environment settles all doubts about the and end up in surface waters receiving the contaminated wastewa- ubiquitous presence of these chemicals in water streams. They ter efﬂuents. Aquatic ecosystems are important targets of pollutantpharmaceuticals as these organisms are exposed to these com-pounds over their whole life via wastewater residues (More over, pharmaceuticals are designed to have speciﬁc ⇑ Corresponding author. Tel.: +36 1 2102930; fax: +36 1 3036968.
mode of action and many of them for persistence in the organism.
E-mail address: (L. K} 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
ohidai / Chemosphere 89 (2012) 592–601 The occurrence pattern and the concentration of the wide range metals, insecticides) compared to bacterial tests such as MicroTox of pharmaceutically active compounds largely vary from country test ) or to a human hepatoma cell line based to country. Nevertheless, the most frequently detected prescription cytotoxicity assay ). A direct comparison of the classes include non-steroidal antiinﬂammatory drugs (NSAIDs), change in viability by Tetrahymena and ﬁsh cells in response to antibiotics, beta-adrenergic antagonists (b-blockers) and iodinated heavy metals showed some very similar responses as well as some X-ray contrast media. The observed levels of these aquatic contam- differences (The interspecies correlation study inants ) that range from the low ng L1–lg L1 are prog- of showed a poor interspecies correlation be- nosticated to increase as the consumption of these drugs is tween T. pyriformis, algae and D. magna for organic chemicals predicted to rise further ( which suggests a high level diversity in the uptake and toxic mech- The environmental risk assessment of these compounds is anisms and underlines the importance of the simultaneous use of encumbered by several difﬁculties such as the lack of ecotoxicity different species in ecotoxicity assessments ().
data especially chronic ones and measuring tools that are able to Beside growth impairment tests, Tetrahymena behavioral assays detect and monitor the presence of pharmaceuticals in surface studying migratory responses of this motile organism are also waters. Nevertheless the range of the available ecotoxicity data is common. A very broad range of biologically active natural often overlapping with the detected environmental concentrations compounds e.g. hormones (or anthropo- as in the case of diclofenac, ibuprofen, paracetamol and sulfameth- genic chemicals including pentachlorophenol, opiod drugs and oxazole especially if these compounds are present in mixtures chloramphenicol ) were described to inﬂuence Tetrahymena migratory behavior in sublethal, nanomolar concen- The complex exposure situations caused by the aquatic pollu- trations. On the basis of the above described advantages of the tants can be in general realistically described by the concept of Tetrahymena model, in the present work our aim was concentration addition as most of the contaminants are nonpolar (i) to screen the toxicity of fourteen human pharmaceuticals organic compounds that show no speciﬁc mode of action and () that belong to the most frequently detected pre- whose toxicity is governed by hydrophobicity ( scription classes (anti-inﬂammatory and analgesic drugs, This concept implies that substances applied at low or no effect antibiotics, b-blockers and X-ray contrast media) to the T.
concentration can contribute to the total mixture effect which in turn can become signiﬁcant. Concentration addition was observed (ii) to describe the type of interactions of the most toxic phar- e.g. for NSAIDs in Daphnia and algal bioassays by maceuticals in combinations (additivity, synergism or .Tetrahymena pyriformis is a non-pathogenic freshwater cili- ate protozoan the abundance of which may indicate healthy aqua- (iii) to study the inﬂuence of sublethal, ecotoxicologically rele- tic environments. It represents an important trophic level where vant concentrations of drugs on the chemotactic responses bioaccumulation processes may be signiﬁcant of the model organism.
). As a microbial fauna member at WWTPs, this species wasfound to ameliorate the stability and performance of biologicalwastewater treatment ). Besides practicaladvantages such as short generation time (150 min) which 2. Materials and methods allows a relatively large number of generations (10) to be studiedeven during 24 h of exposure, the use of Tetrahymena may also be 2.1. Cell culturing favorable because it shows a lot of similarities with higher rankedvertebrates in terms of e.g. receptors } Tetrahymena pyriformis GL cells were maintained in 0.1% yeast and second messenger systems ( extract containing 1% Bacto tryptone (Difco, Michigan, USA) med- Tetrahymena growth impairment tests were reported to show ium at 28 °C. For both the chemotaxis and the growth inhibition signiﬁcantly higher sensitivity to diverse xenobiotics (e.g. heavy assays 24 h old, exponential growth phase cultures were used.
Table 1Mechanism of action, logarithmized partitioning coefﬁcient (log Kow) and environmental concentrations of the pharmaceuticals tested.
Environmental concentration (ng L1) Acetylsalicylic acid NSAID; nonselective inhibition of COX-2 Analgesic-antipyretic; inhibition of COX-3? Bacteriostatic; inhibition of the 50S ribosome <287 (Kümmerer, 2009) 4.5 (Zuccato et al., 2005) subunit during translation 24.4 (Zuccato et al., 2005) <730 (Kümmerer, 2009) Bacteriostatic; inhibition of the bacterial dihydropteroate syntethase involved in DNAreplication Bacteriostatic; inhibition of the bacterial dihydrofolate reductase involved in DNAreplication Blocking of the b-adrenergic receptor 5.5 (Gabet-Giraud et al., 2010) X – ray contrast agent 2 (Putschew et al., 2000) * estimated partitioning coefﬁcients of the non-ionized compounds, retrieved from the database of PubChem project (http://pubchem.ncbi.nlm.nih.gov/).
** COX: Cyclooxygenase.
ohidai / Chemosphere 89 (2012) 592–601 2.2. Pharmaceuticals The positive responder cells detect developing concentration gra-dients in the microscopic proximity of capillary openings and this In both chemotaxis and single compound toxicity assays 14 advantageous chemical signal guides chemotactically active cells human pharmaceuticals were tested including ﬁve NSAIDs, one into the upper chamber, while negative responders migrate away.
antipyretic–analgesic drug, four antibiotics, three b-blockers and Our previous experiments has proved that taking short incubations an X-ray contrast agent All compounds were obtained times (5–20 min) for the assay facilitate to gain pure gradient from Sigma Aldrich, Germany. Water soluble drugs (diclofenac- directed chemotactic responses and prevent the contamination of Na, ibuprofen-Na, naproxen-Na, lincomycin hydrochloride, meto- the samples from randomly running chemokinetic responder cells (Schiess et al., 2001). Following 20 min of incubation positive Na-diatrizoate) were diluted in the culture media; whereas hydro- responder cells were ﬁxed and counted with the CASYÒ TT cell phobic substances (acetylsalicylic acid, fenoprofen-Ca, paracetamol, counter system. The tested drug concentration varied from erythromycin, sulfamethoxazole, trimethoprim) were diluted in 1015 M to 106 M and each measurement was done in 12 replicas.
DMSO. The concentration of stock solutions was uniformly Chemoattractant or repellent effect of pharmaceuticals was quan- 0.05 M, the consecutive dilutions of the stock solutions were made tiﬁed by the ‘Chemotaxis Index' (Chtx. Ind.) which was calculated as in culture media. For the sake of simplicity pharmaceuticals will be the ratio of the number of cells that migrated towards the chamber referred hereinafter as the name of the active agent.
containing the test substance and the number of cells thatmigrated towards the chamber containing the reference culture 2.3. Growth inhibition assay of single pharmaceuticals and mixtures media as mentioned in Section .
For all toxicity tests 96-well plates (Sarstedt, Germany) were 2.5. Statistical analysis and curve ﬁtting seeded with 103 cells well1. Then the cells were exposed to singlepharmaceuticals or binary combinations of them for 24 h. For each In both chemotaxis assay and growth inhibition assay statistical measurement points at least 18 replicas were done. Following signiﬁcance of the results compared to the respective controls was ﬁxation with PBS containing 4% formaldehyde the cell number calculated with one-way analysis of variance (ANOVA) in the was determined using the CASYÒ TT cell counter system (Innova- Origin7.0. In mixture toxicity assay the obtained growth inhibition tis-Roche, Switzerland). The function of the equipment is based effects of mixtures were compared to the sum of the individual on the electric current exclusion principle. The cell number and growth inhibition effects of the two mixture constituents and the size are registered based on the number and amplitude of these signiﬁcance of the difference was evaluated by one-way ANOVA.
electrical signals, respectively. This means that when cells pass Interaction of two drugs was considered as a ‘simple additivity' if through a pair of electrode they increase resistance due to their the observed mixture toxicity did not differ signiﬁcantly from the insulating plasma membrane. For Tetrahymena the considered sum of the individual toxic effects. If the growth inhibition exerted particle diameter ranged from 10 to 100 lm.
by the mixture of the two components was signiﬁcantly higher Growth inhibition (%) was calculated as the ratio of the average than the sum of their individual toxic effects it was described as cell number in the sample wells and the average cell number in ‘synergism'. On the contrary, interaction was considered to be an control wells. For hydrophilic drugs control wells contained pure ‘antagonism' if the combined toxic effect was signiﬁcantly lower culture media, whereas for hydrophobic ones culture media con- than the sum of the individual toxicity values. In the case of the taining the adequate volume ratio of DMSO served as a control.
four selected pharmaceuticals (see Section ) individual concen- In the single compound growth inhibition assays that aimed to tration-growth inhibition curves were established. Based on the identify the most toxic pharmaceuticals the concentration range assumption of a monocausal relationship between concentration was tested in 1011–103 M using a 10-fold dilution between each and response, symmetric sigmoidal curves were ﬁtted to measure- measurement point. In the second step of the test the two most ment data (8 measurement points for each pharmaceutical with 18 toxic hydrophilic members of the two most potent growth inhibi- replicas at each point) in the Origin7.0. For the sigmoidal curve tor prescription classes were selected to be tested in binary ﬁtting a four parameter logistic function was used by the following mixtures. For this purpose ﬁrst their individual concentration- growth inhibition curves were established using concentrationlevels determined by preceding range-ﬁnding tests. For the details of the curve ﬁtting see Section Binary mixtures were prepared from the four selected drugs in all the six possible combinations. The toxicity of these mixtures was where A1 upper asymptote represents the maximal proliferation studied using the concept of concentration addition inhibition (about 100%); A2 lower asymptote stands for the minimal and the Toxic Unit approach () that expresses proliferation inhibition (about 0%); x0 represents the concentration the concentration of each mixture constituent as the ratio of its ac- at which proliferation inhibition equals 50% (the EC50 value) and p tual concentration and its EC stands for the slope. The standard errors of the parameters obtained 50 value. Concentrations of mixture constituents were set according to a 24 full factorial design in which were also evaluated. For each pharmaceutical the individual toxic individual factor levels corresponded to 0.25, 0.50, 0.75 and 1.0 Toxic effects at the concentrations representing 25%, 50%, 75% and 100% of the respective EC50 values were determined using the ﬁttedcurves.
2.4. Chemotaxis assay The measurement of the chemotactic responses induced by the 14 pharmaceuticals took place in a two chamber multichannel 3.1. Growth inhibiting effects of single pharmaceuticals capillary assay described by at 28 °C and at normal lighting conditions. For this purpose cells were placed in a 96-well Toxicity screening of the 14 pharmaceuticals showed that plate (lower chamber) at 105 cells well1 density whereas test sub- NSAIDs and b-blockers were the most toxic classes to T. pyriformis stances were ﬁlled in the tips of a multichannel pipette (upper as all of the ﬁve NSAIDs and three b-blockers exhibited signiﬁcant chambers) placed above the wells containing the cell suspension.
growth inhibiting effect in the 1011–103 M range ( ohidai / Chemosphere 89 (2012) 592–601 Table 2Concentration dependent impact on growth of the fourteen drugs tested in the ﬁrst toxicity screening.
Effect on growth (%)⁄ Acetylsalicylic acid ⁄ ‘‘ + '' values correspond to growth enhancement; ‘‘'' values represent growth inhibition. Signiﬁcant effects are written in bold. Signiﬁcance levels are x: p < 0.05; y: p < 0.01;z: p < 0.001.
However, except for acetyl salicylic acid these drugs inhibited cell 50%, 75% and 100% of the respective EC50 values were predicted proliferation only in the 105–103 M concentrations (growth inhi- base on the ﬁtted curves and checked by growth inhibition assay.
bition ranged from 12.4 ± 1.9% for 105 M ibuprofen to 94.2 ± 0.8% The difference of predicted and measured data was not signiﬁcant for propranolol at 103 M) which are at least three order of magni- in any case. Respective predicted values were 14%, 29%, 41% and tude higher than the environmental concentrations. On the con- 50% for diclofenac, whereas measured effects were 15.9 ± 5.7%, trary, acetyl salicylic acid had signiﬁcant but less pronounced 30.4 ± 5.5%, 42.2 ± 3.7% and 48.3 ± 1.9%. In the case of ibuprofen effect (16.6 ± 6.0%–26.0 ± 3.5%) in the 1011–108 M range. Hence predicted effect values were 10%, 25%, 39%, 50% and measured for NSAIDs the increasing order of toxicity based on the prolifera- growth inhibitions were 14.6 ± 4.8%, 33.1 ± 8.1%, 41.9 ± 5.9% and tion inhibition at the respective lowest effective concentrations 47.1 ± 3.1%. For metoprolol predicted data were 20%, 33%, 43% was naproxen < ibuprofen < diclofenac < fenoprofen < acetyl sali- and 50%, on the other hand measurement data were 19.4 ± 10.1%, cylic acid. The antipyretic–analgesic paracetamol in turn signiﬁ- 35.3 ± 8.2%, 37.5 ± 9.8% and 49.9 ± 6.3%. Finally, for propranolol cantly promoted cell proliferation with 42.0 ± 11.5% at 103 M predicted growth inhibitions were 10%, 25%, 39% and 50% while and similar but weaker effect was also observed in the entire con- measured data were 14.8 ± 8.5%, 23.1 ± 9.5%, 36.3 ± 13% and centration range. From b-blockers propranolol proved to be the 46.8 ± 7.3%.
most potent as it showed 13.5 ± 3.5% growth inhibition at 106 Mwhereas signiﬁcant growth inhibiting effect of metoprolol and 3.2. Toxicity of binary mixtures timolol was observed above 105 M and 104 M respectively. Formetoprolol growth inhibition increased from 12.4 ± 4.0% to Combined effects of diclofenac, ibuprofen, metoprolol and 82.0 ± 2.4% with the rising concentrations. In the case of timolol propranolol were studied in all of the six possible binary mixtures.
growth inhibiting effects were 29.9 ± 4.9% at 104 M and 75.9 ± Observed mixture proliferation inhibiting effects were compared 1.3% at 103 M. Consequently the observed order of toxicity was to the sum of the individual proliferation inhibitions of the two timolol < metoprolol < propranolol.
mixture constituents at their respective actual concentrations in Sulfamethoxazole and lincomycin did not affected the cell pro- order to determine the type of interaction, i.e. additivity, syner- liferation signiﬁcantly at any concentration assayed, whereas gism, antagonism (as described in Section erythromycin had proliferation promoting effect at 1011–105 Using the diclofenac + ibuprofen mixtures additivity occurred M, particularly in the 1010–108 M range where proliferation only in the mixture containing the lowest concentration of both dic- was enhanced with 25.4 ± 5.6%, 19.6 ± 3.6% and 13.4 ± 4.9% respec- lofenac and ibuprofen (0.25 TU diclofenac + 0.25 TU ibuprofen) tively. The iodinated X-ray contrast agent Na-diatrizoate showed while at all other concentrations antagonism was the observed type no proliferation inhibition at any concentration, on the contrary of interaction In the diclofenac + metoprolol it had slight proliferation enhancing effect in the entire concentra- combinations additivity was the predominant form of combined tion range (that did not exceed 16.0 ± 2.7%).
action, synergism was observed in the case of the mixture 1 TU Based on the results listed above diclofenac, ibuprofen, meto- diclofenac + 0.25 TU metoprolol and with the three mixtures of prolol and propranolol were chosen for the study of combined ef- 1.5 TU total concentration (0.5 TU diclofenac + 1 TU metoprolol; fects in binary mixtures. This selection was made using three 0.75 TU diclofenac + 0.75 TU metoprolol; 1 TU diclofenac + 0.5 TU criteria: (i) the most toxic, (ii) water soluble pharmaceuticals were metoprolol) Antagonism was observed at selected, (iii) two compounds from two different prescription clas- the highest concentration (1 TU diclofenac + 1 TU metoprolol).
ses so that combined effects of pharmaceuticals with both similar Using the diclofenac + propranolol mixtures additivity was observed and different mode of actions could be studied. As a ﬁrst step indi- in seven cases and antagonism was observed in nine cases vidual concentration–growth inhibition curves were established (Interestingly, the two interaction types for these compounds ). It was found that EC50 values were alternated. At the two lowest mixture concentrations (0.5 TU and 8.35 105 ± 7.09 106 M (26.56 ± 2.26 mg L1) for diclofenac, 0.75 TU) additivity was observed whereas at intermediate mixture 2.05 104 ± 7.81 106 M (46.79 ± 1.78 mg L1) for ibuprofen, concentration (1 TU, 1.25 TU and 1.5 TU) both additivity and 4.51 104 ± 2.30 105 M (308.85 ± 15.75 mg L1) for metopro- antagonism was observed. At the highest mixture concentrations lol and 1.63 105 ± 9.45 107 M (4.82 ± 0.28 mg L1) for pro- (1.75 TU and 2 TU) the interaction type was antagonism. Similarly, pranolol. Growth inhibition effects corresponding to the 25%, with the ibuprofen + metoprolol mixtures at the lower mixtures
ohidai / Chemosphere 89 (2012) 592–601 Fig. 1. Fitted concentration–growth inhibition curves and the respective ﬁtting parameters of diclofenac, ibuprofen, metoprolol and propranolol. These four potent growthinhibitor drugs were selected for the binary mixture studies (see Section and Appendix A).
concentrations (0.5 TU and 0.75 TU mixture) additivity was 1014 M and 1011–109 M; the respective Chtx. Ind. ranged from observed while at the higher mixture concentrations (1.5 TU, 1.75 73.8 ± 3.6% to 87.0 ± 2.9%. Fenoprofen also showed repellent TU and 2 TU) antagonism was found ).
character at 1014 M (Chtx. Ind = 70.5 ± 9.2%) and ibuprofen was At the intermediate concentrations (1 TU and 1.25 TU) antago- repellent at 107 M (Chtx. Ind = 66.3 ± 6.1%). Similarly, the analgesic nism was predominant, but additivity was also observed. The paracetamol was repellent at 1014–1011 M (Chtx. Ind = 77.2 ± ibuprofen + propranolol mixtures showed similar interaction proﬁle 4.4%–84.8 ± 2.8%) yet at 1013 M this effect was not signiﬁcant.
to the ibuprofen + metoprolol combination except that additivity Naproxen in turn was the only anti-inﬂammatory drug that showed was more frequent at the intermediate mixture concentrations a weak, however, signiﬁcantly chemoattractant effect at 1013 M (1 TU and 1.25 TU) and antagonism occurred also at low mixture (Chtx. Ind = 127.3 ± 10.1%).
concentrations (0.5 TU and 0.75 TU) Finally, Three of the four antibiotics exhibited signiﬁcant chemoattrac- with the metoprolol + ibuprofen mixtures antagonisms were tant character erythromycin at 108 M (145.9 ± 18.3%); observed in almost all cases similarly to the described one for the lincomycin at 1015 and 109 M (Chtx. Ind = 170.3 ± 20.8% and diclofenac + ibuprofen combination except for three mixtures 133.3 ± 9.2%); sulfamethoxazole at 1011 M, 109 M and at (0.25 TU metoprolol + 0.25 TU propranolol; 0.5 TU metoprolol + 106 M (Chtx. Ind = 125.6 ± 9.2%–146.1 ± 20.3%). However, eryth- 0.25 TU propranolol; 0.5 TU metoprolol + 0.5 TU propranolol) romycin and lincomycin had also repellent actions: erythromycin where additivity was observed ().
was repellent at 1015 (Chtx. Ind = 72.1 ± 10.2%) whereas lincomy-cin was repellent at 106 M (Chtx. Ind = 78.3 ± 9.9%). Trimethoprim 3.3. Chemotactic effects of single pharmaceuticals at sublethal was neutral in the entire concentration range.
Among b-blockers () metoprolol was repellent at 1015 M, 1014 M, 107 M and 106 M (Chtx. Ind = 67.0 ± 5.5%–82.3 ± 7.5%).
Migratory responses of T. pyriformis elicited by the fourteen Propranolol showed also repellent effects at 1015 M. 1011 M pharmaceuticals were tested in sublethal concentrations in the and 1010 M (Chtx. Ind = 62.8 ± 2.0%–71.3 ± 4.2%) whereas timolol 1015–106 M range that covered environmentally relevant concen- acted in similar way at 1015 and 1014 M (Chtx. Ind = 67.0 ± trations (below 109 M). It was found that with the exception of na- 10.5% and 75.1 ± 11.5% respectively) and had attractant character proxen all anti-inﬂammatory–analgesic pharmaceuticals exhibited at 106 M (Chtx. Ind = 150.0 ± 13.3%). The iodinated X-ray contrast signiﬁcant chemorepellent character ). Acetyl salicylic acid media Na-diatrizoate had also ambiguous, concentration depen- had weak repellent action at 1015 M (Chtx. Ind. = 84.2 ± 3.4%), dent character since it was attractant at 1014 M, 107 M and 109 M (Chtx. Ind. = 80.4 ± 8.5%) and at 106 M (83.9 ± 7.6%).
106 M (Chtx. Ind = 137.5 ± 14.1%–209.3 ± 20.1%) and it was repel- Diclofenac acted as a weak chemorepellent in a wide concentration lent at 1013 M and 108 M (Chtx. Ind = 83.7 ± 4.4% and 64.3 ± range from 1015 M to 109 M. This effect was signiﬁcant at 1015 M,
ohidai / Chemosphere 89 (2012) 592–601 Fig. 2. Chemotactic responses of Tetrahymena cells induced by the ﬁve anti-inﬂammatory drugs acetyl salicylic acid, diclofenac, fenoprofen, ibuprofen, naproxen and theanalgesic paracetamol. Signiﬁcance level is x: p < 0.05.
(log Kow = 1.9). This observation was consistent with the ﬁnding of(who reported in a QSAR approach based study 4.1. Growth inhibiting effects of single pharmaceuticals that NSAIDs acted on Daphnia and algae by nonpolar narcosisand that the higher the log Kow of the substance the higher was Screening of the growth inhibiting effect of the 14 pharmaceu- its toxicity. However, in both the work of Cleuvers and the present ticals showed that Tetrahymena pyriformis was the most sensitive study results were obtained using salt forms of the pharmaceuti- to treatment with b-blockers and NSAIDs. However, our results cals. Furthermore in an interspecies correlation experiment show that the observed effective concentrations are at least three ) a poor interspecies correlation were found for hydro- orders of magnitude higher than environmental concentrations phobic substances between Tetrahymena and Daphnia or algae suggesting that acute toxic effects of these pharmaceuticals are suggesting that depending on the organic chemical modes of up- improbable in the aquatic environment. Obtained EC take and action might differ from species to species ( the four selected drugs: 26.56 mg L1 for diclofenac, 46.78 mg L1 Moreover, the establishment of quantitative relationship for ibuprofen, 608.85 mg L1 for metoprolol and 4.82 mg L1 for between certain molecular descriptor physicochemical parameters propranolol are comparable to literature data such as the study and biological effect (toxicity) may not be possible for all kinds of of which reported that the sensitivity of Daphina chemicals. In their work () for example found immobilization test, Lemna and Desmodesmus algal growth inhibi- weak correlation between the toxicity and the log Kow (R2 = 0.59) tion tests were in the same range (EC value of organic contaminants such as pesticides, insecticides, 50 values varied between 5 mg L1 and >320 mg L1). The reported order of toxicity diclofe- and combustion products. Hence the application of QSAR approach nac > ibuprofen > naproxen and propranolol > metoprolol was also might depend on the types of chemicals. Nevertheless, the other identical to what was found by us. Comparison of this order anti-inﬂammatory–analgesic pharmaceuticals seemed to ‘‘obey'' with the most frequently evoked molecular descriptor related to to the above mentioned rule i.e. higher log Kow means higher toxic- the biodistribution and toxic effect of organic chemicals, the ity. Fenoprofen having a relatively high log Kow value of 3.3 showed logarithmized octanol–water partitioning coefﬁcient ), elevated toxicity whereas paracetamol possessing a low log Kow shows that in our experiment toxicity increased together with value of 0.5 had no proliferation inhibiting effect at all. Similarly, ow: diclofenac (log Kow = 4.4) > ibuprofen (log Kow = 3.5) > b-blocker timolol with a log Kow = 1.8 which is very close to that of metoprolol (log K ow = 3.3) and propranolol (log Kow = 3.0) > metoprolol ow = 1.9), was almost as potent
ohidai / Chemosphere 89 (2012) 592–601 Fig. 3. Chemotactic responses of Tetrahymena cells elicited by the antibiotics: erythromycin, lincomycin, sulfamethoxazole and trimethoprim. Signiﬁcance levels are x:p < 0.05 and y: p < 0.01.
proliferation inhibitor as the metoprolol. An interesting behavior, the prokaryotic ribosome (). The two other antibiotics weak proliferation inhibition (20%) at low concentrations sulfamethoxazole (log Kow = 0.9) and trimethoprim (log Kow = 0.9) (1011–108 M) but not at high concentrations was observed with in turn exhibited weak proliferation inhibition effect in higher acetyl salicylic acid (log Kow = 1.2) which was described by previ- concentrations (107–103 M). Originally, both of them are bacte- ous studies to inhibit Tetrahymena growth in high (104 M) con- riostatic due to their ability to abolish bacterial folate synthesis centration (Different results may be via the inhibition of the dihydropteroate syntethase and the attributed to the diverse experimental setup (such as cell density, dihydrofolate reductase respectively. However, even though with culture volume and media).
a much lower (about 104 fold weaker) afﬁnity they are also able In Tetrahymena, however, speciﬁc mode of action of the NSAIDs to interact with homologous eukaryotic targets (such and b-blockers might not be excluded either since it posses the as the bifunctional dihydrofolate reductase-thymidylate synthase molecular target of both classes i.e. the b-adrenergic receptor of Tetrahymena which has already been described not only from (and cyclooxygenase (However, a functional point of view ) but also at the geno- genomic data are not yet available in the Tetrahymena Genome mic level (the Tetrahymena Genome Database gene identiﬁer is Database ) to compare them with the hu- TTHERM_00312120). Protein BLAST alignment of the sequence re- man homologues in order to draw some conclusions about their veals that it both the dihydrofolate reductase and the thymidylate structure and afﬁnity towards the human pharmaceuticals. For synthase part contain several putative conserved domains. Homol- propranolol it is also known that its higher toxicity compared to ogous proteins were found in species at different levels of phylog- other b-blockers is due to its strong membrane stabilizer potency eny such as plants (e.g. Hordeum vulgarea, the common barely) as that the other b-blockers lack ( well as in higher ranked vertebrates such as mice or human.
The low sensitivity of Tetrahymena to antibiotics might be The X-ray contrast agent Na-diatrizoate (log Kow = 1.8) did not attributed to the fact that they are designed to act speciﬁcally on elicit proliferation inhibition either. Though little is known about bacterial targets sites (This might be the reason why eryth- the possible toxic effects of iodinated contrast media, a study using romycin with the highest octanol–water partitioning coefﬁcient multiple biomarkers including phospholipids and adenosine (log Kow = 2.7) together with lincomycin (log Kow = 0.2) did not show triphosphate (ATP) reported that neither Na-diatrizoate nor its proliferation inhibiting potency at all. In fact both substances metabolites affected bacteria present in the sewage sludge inhibit bacterial protein synthesis via interfering speciﬁcally with
ohidai / Chemosphere 89 (2012) 592–601 Fig. 4. Chemotactic responses of Tetrahymena cells induced by the b-blockers metoprolol, propranolol, timolol and the iodinated X-ray contrast media Na-diatrizoate.
Signiﬁcance levels are x: p < 0.05 and y: p < 0.01.
4.2. Combined action of diclofenac, ibuprofen, metoprolol and and the observed interaction type as it was suggested for other propranolol in binary mixtures contaminants such as heavy metals (but nocorrelation was found. The complicated concentration dependent Predominant interaction type observed in mixtures was antag- interaction type pattern seems to better corroborate with the onism (in 59% of the combinations) and the frequency of its detec- ﬁndings who studied the interactions of 13 tion increased in general with the mixture concentration. It was environmentally relevant pharmaceuticals (such as ibuprofen, car- found in all combinations with mixture concentration above 1.25 bamazepine, and sulfamethoxazole) and found complex concen- TU (except for the combination 1 TU diclofenac + 0.5 TU proprano- tration dependent interaction proﬁle difﬁcult to predict or model.
lol and the diclofenac + metoprolol mixtures). The predominanceof antagonism in the higher concentration range might be ex- 4.3. Chemotactic responses of Tetrahymena to environmental plained by a potential competitive inhibition between the two concentrations of pharmaceuticals pharmaceuticals acting on the same molecular target. However,it is more difﬁcult to give an explanation to antagonisms observed Chemotaxis is a highly sensitive cell physiological response that between pharmaceuticals having different molecular targets. Any- can be elicited by a large variety of biologically active compounds at how, our results suggest that the concept of concentration addition low concentrations. In environmental toxicology it was mainly validated for the NSAIDs in algal and Daphnia biotests studied in the context of bioremediation of polluted soil or ground ) cannot be fully adopted for Tetrahymena population inhibi- waters. It was found that the chemotaxis of soil inhabiting micro- tion assay. This may be due to the interspecies differences in the bial community members can enhance biodegradation of pollu- uptake and mechanism of action of these pharmaceuticals in these tants (e.g. aromatic hydrocarbons) by overcoming limitations in model organisms that was also stressed by As a the bioavailability of these later Our matter of fact, concentration additivity was observed only in the results show that 13 of the tested pharmaceuticals altered signiﬁ- 37% of the mixtures. Synergism was the rarest type of interaction cantly chemotactic responses of Tetrahymena in sublethal concen- that was obtained only in 4% of the combinations and only in trations with trimethoprim being the only exception that were diclofenac + metoprolol mixtures (at relatively high mixture con- neutral. The respective effective concentrations were in the range centrations of 1.25 and 1.5 TU).
of the environmental concentrations (<109 M) except for the che- In order to explain the observed interaction type pattern we morepellent effect of ibuprofen. The chemotactic proﬁle of the compared the concentration ratio of the two mixtures constituents pharmaceuticals was not strictly correlated to their toxicity. The ohidai / Chemosphere 89 (2012) 592–601 most toxic drugs (diclofenac, metoprolol, propranolol) tended to of this model can be ﬁnely and selectively modulated by pollutants have chemorepellent effect, but important differences could be ob- and their mixtures. Furthermore the chemotactic responsiveness served in terms of effective concentration and the wideness of the should be also considered as a signiﬁcant evaluation criteria in chemorepellent concentration range. Moreover some pharmaceuti- pollution assays. However, the use of some speciﬁc biochemical cals such as timolol that proved to be quite toxic had not only che- endpoints could also help to better understand the mechanism of morepellent but also attractant effect. Paracetamol, erythromycin the action of aquatic contaminant pharmaceuticals in Tetrahymena and lincomycin in turn, that did not inhibit proliferation exhibited as well as in protozoan level in general.
Literature data about the chemotactic effect of these pharma- Appendix A. Supplementary material ceuticals were mostly obtained in chemotaxis inhibition experi-ments where interaction of these drugs with the function of the Supplementary data associated with this article can be found, immune system was studied using chemoattractant stimulated in the online version, at human polymorphonuclear leukocytes (PMNs). Diclofenac and ibu- profen were described to reduce chemotaxis of PMN and monocytecells induced by potent chemoattractants like the substance P, transforming growth factor-b ) and the bacte-rial tripeptide fMLF Similarly, the b-blockers Berenbaum, M.C., 1985. The expected effect of a combination of agents: the general metoprolol and propranolol but not timolol decreased the fMLF in- solution. J. Theor. Biol. 114, 413–431.
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