Introduction: General concepts

The term biomarker was originally defined as “a detectable biochemical or physiological alter-ation or cellular manifestation brought about byenvironmental stress”. In the present context, theconcept of a biomarker is used in a more restrictivesense, namely “a sub-lethal biochemical change re-sulting from individual exposure to a toxic pollu-tant” (Gadd 1992). Therefore, any cellular systemwhich undergoes a detectable physiological alter-ation under the influence of a specific environmen-tal stress or pollutant can be considered as a cellbiomarker, and molecules involved in this alter-ation or physiological change may be consideredmolecular biomarkers (e.g. metallothionein bio-synthesis under heavy metal exposure).

On the other hand, the classical biosensor con-cept involves the existence of two components; abioreceptor (biological material) and a physico-

Ciliates as a potential source of cellular and molecularbiomarkers/biosensors for heavy metal pollution

Juan Carlos Gutiérrez*, Ana Martín-González, Silvia Díaz and Ruth Ortega

Departamento de Microbiología-III, Facultad de Biología, C/. José Antonio Novais, 2,Universidad Complutense (UCM), 28040 Madrid, Spain; E-mail: jgf00004@teleline.es

Received: 2 September 2003; 9 October 2003. Accepted: 13 October 2003

Ciliates can be valuable eukaryotic micro-organisms for use as whole-cell biosensors or as a potentialcellular source of molecular biomarkers/biosensors to detect pollutants (such as heavy metals) in envi-ronmental samples. Here, we report the advantages of using ciliates in biomonitoring of heavy metals, incomparison with other micro-organisms. The diversity of experimental conditions and methodologicalapproaches in heavy metal bioassays using ciliates are also discussed. Finally, we show several examplesof the suitability of ciliates as potential whole-cell or molecular biosensors to detect bioavailable heavymetals in environmental samples.

Key words: Biomarkers; Biosensors; Ciliates; Fluorophores; Heavy metals; Metallothioneins.

chemical transducer. The bioreceptor might be abiomolecule or a whole cell that recognises the tar-get (heavy metal), whereas the transducer convertsthe recognition event into a measurable signal. Thesensing elements (bioreceptors) might be enzymes,antibodies, DNA molecules, cell receptors and or-ganelles, or whole cells of micro-organisms as wellas of animals or plants, and the transducer may beelectrochemical or mechanical, optical or acoustic.The function of a biosensor depends on the bio-chemical specificity of the biologically active mate-rial. The choice of the biological material will de-pend on a number of factors, including the natureof the pollutant to be detected.

Recently, the concept of the whole-cell biosen-sor has been introduced by several authors(D’Souza 2001; Belkin 2003), as a very useful al-ternative to classical biosensors. A whole-cellbiosensor uses the whole prokaryotic or eukary-otic cell as a single reporter incorporating both

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*corresponding author

Europ. J. Protistol. 39, 461–467 (2003)© Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/ejp

bioreceptor and transducer elements. In general,living systems to be used as whole-cell biosensorsare experimentally modified to incorporate thetransducer capacity.

In environmental biomonitoring, global param-eters such as bioavailability, toxicity and genotoxi-city can not be tested using molecular recognitionor chemical analysis, but can only be assayed usingwhole cells. Obviously, in these bioassays thequestion to be resolved is not “What toxicantsdoes the sample contain?”, but rather “How toxicis the sample?”

Two types of bioassays using whole-cell biosen-sors may be considered: “turn off” and “turn on”assays. In “turn off” assays (which are similar togeneral microbial toxicological bioassays), thesample toxicity is estimated from the degree of in-hibition of a cellular activity that is a normallycontinuous (e.g. inhibition of growth, respirationor metabolism, motility or the biosynthesis of aspecific molecule), and is based on the measure-ment of a decrease in growth rate, light (fluores-cence/bioluminescence) emission, colour-less cellpopulation, motility, etc. as a function of sampletoxic concentration. For instance, a good exampleof a “turn off” assay using ciliates, may be a report-ed rapid bioassay to detect mycotoxins using amelanin precursor overproducer mutant of the cil-iate Tetrahymena thermophila (Martín-Gonzálezet al. 1997). On the other hand, in “turn on” assaysa quantifiable molecular reporter is fused to a spe-cific gene promoter (like a metallothionein pro-moter), known to be activated by the target chemi-cal or environmental pollutant (such as a heavymetal).

Advantages in using micro-organismsas whole-cell biosensors/biomarkersin environmental pollution monitoring

As indicated above, biological systems, particu-larly micro-organisms, provide the best ways to as-sess pollutant bioavailability. This ecotoxicologicalparameter is really important in heavy metal pollu-tion because metal ions can bind to abiotic surfacesand ligands (like particulate organic matter, clayand metal (hydroxides/oxides), which may strong-ly influence their bioavailability and, therefore,their cellular toxic effects. For this reason the or-ganic matter content and other factors (like pH) of

the culture medium are important parameters to beconsidered in any heavy metal toxicological bioas-say. Furthermore, the use of micro-organisms pro-vides data on the biological effects following expo-sure to heavy metals, which may be extended toother more complex living systems. Also, they canprovide data on heavy metal interactions (synergis-tic or antagonistic toxic effects) in metal mixtures.Heavy metal pollution is usually multiple (two ormore different metals are typically present), and thecellular toxic effect of a metal can vary in responseto the presence of other heavy metals. For instance,calcium and zinc have an antagonistic effect, likecadmium and zinc, whereas mercury increases thetoxicity to zinc (Nies 1999). We have observed inthe soil ciliate Colpoda steinii that the mediumlethal concentration (LC50) of cadmium increasesabout 15 times in the presence of zinc at sub-lethalconcentrations (unpublished work).

In general, micro-organisms show a rapidgrowth rate, reaching a large cell population in ashort time, which is important in some ecotoxico-logical assays to obtain an amplified response fordetecting the pollutant-induced change, whenmicro-organisms are used as whole-cell biomark-ers or biosensors. Besides, it is possible to use ge-netically engineered micro-organisms, which arevery useful in “turn on” assays, and to make up ap-propriate genetic constructions to detect a specificpollutant (e.g. heavy metal-specific biosensors).Likewise, specific mutants or micro-organismswith several gene reporters might be used. Geno-toxicity studies are simpler and more practicableusing micro-organisms because they can more eas-ily be subjected to genetic dissection.

Additional advantages of using ciliatesas whole-cell biomarkers/biosensors

Ciliates are eukaryotic micro-organisms; theyhave all the advantages previously reported, andthey present at least two additional advantages:

a) In contrast to bacteria and yeasts, ciliates areunicellular organisms without a cell wall in the veg-etative stage. A major limitation to the use of bacte-ria or yeasts as whole-cell biosensors is the uncer-tainty concerning diffusion of substrates and prod-ucts through the cell wall, which may result in aslower or less effective response as compared to en-zyme-based sensors. To obviate this problem suchcells must be permeabilised using physico-chemical

462 J. C. Gutièrrez et al.

or enzymatic methods to allow the free diffusion ofsmall molecules across the cell membrane, but thecells are no longer normal viable organisms. Theuse of ciliates, instead of bacteria or yeasts, mightavoid this serious problem. The absence of a cellwall in these eukaryotic micro-organisms mightoffer a higher sensitivity to environmental pollu-tants, and, therefore, a faster cellular response.

b) They are eukaryotic micro-organisms withsome metabolic traits that are more similar to thoseof human cells than are bacteria or yeasts. Pilotgenome projects in two model ciliates often used inecotoxicological studies, Tetrahymena and Para-mecium (Dessen et al. 2001; Turkewitz et al. 2002),have shown that they share a higher degree of func-tional conservation with human genes than do othermicrobial model eukaryotic micro-organisms. Thisis shown by better matches of relevant ciliate codingsequences to those in humans, as compared withnon-ciliate microbial models. Therefore, it seems tobe more reasonable to use these eukaryotic cells inecotoxicological studies as models for humans, andrepresents an alternative to animal tests.

Diversity of experimental conditionsand methodological approachesin ciliate heavy metal bioassays

To select ciliates in order to design whole-cellbiosensors for heavy metal environmental moni-toring, we should first determine basic ecotoxico-logical parameters (e.g. resistance/tolerance or sen-sitivity to a specific metal) for that ciliate strainunder controlled laboratory conditions. After re-viewing published data on heavy metal ecotoxico-logical analysis using ciliates, we conclude that thediversity of experimental conditions and method-ological approaches is generally too great to makecomparisons between the results using differentciliates. The following points illustrate this gener-alized and serious problem:

a) Heavy metal concentration parameters.Metal concentrations are expressed in diverseforms, generally as weight/volume (ppm or mg/l) ormolarity (µM or mM). If authors report experimen-tal data properly, the conversions among these areeasily possible, but problems arise if concentrationsare expressed in less conventional ways, as a frac-tion of total proteins or of dry weight, for example.

b) Culture medium composition. This canrepresent a significant problem in some circum-

stances, because the concentrations of organic mat-ter and dissolved salts or the presence of sedimentsor clay are not usually reported. These parameters,as well as some pH changes, reduce the heavymetal availability and, therefore, decrease the toxicimpact. For instance, in Tetrahymena pyriformisgrowth is not affected up to a dose of 8 mg l–1 Cd(in 2% proteose-peptone and 0.1% yeast extractmedium) (Sauvant et al. 1999). By contrast, Schae-fer et al. (1994) reported an EC50 at 48 h, of 0.78 mg l–1 Cd (in a medium composed by 1%proteose-peptone and 1% yeast extract) for thesame ciliate. A protective effect of organic matterhas also been observed in other ciliates (Martín-González et al. 1999).

c) Assessment of toxic effect. In general, eco-toxicological assays using ciliates have been con-ducted to assess sub-lethal effects, such as inhibi-tion of growth, variation in feeding rate, inhibitionof respiration, motility, etc.

d) Endpoints. The LD50/LC50 (median lethaldose/median lethal concentration) is the most fre-quently used toxicological endpoint in ciliates, butother measures of physiological effect are alsoused, such as the IG50 (median growth inhibitionconcentration), also named the EC50 (effective con-centration for 50% growth inhibition) or IC50 (me-dian inhibitory concentration), and other moreunusual forms, like LOED/LOEC (lowest ob-served effective dose or concentration) and TLm(median tolerance limit).

e) Duration of heavy metal exposure. In cili-ate toxicological studies the duration of heavymetal exposure used in assays is also very diverse.The majority of authors measure the toxic effect at24 h, but others use very different exposure times,for instance; 1 h (Nilsson 1989), 48 and 72 h(Schaefer et al. 1994), and so on.

f) Cell concentration. This is another very di-verse parameter among ciliate ecotoxicological as-says. The range of cell concentrations is reallylarge; some authors use concentrated cell cultures(e.g. about 103 cells ml–1; Martín-González et al.1997) and others very few cells (e.g. about 12 cells;Madoni et al. 1994). Obviously, LC50 is lower incultures with low cell number, because of the in-crease in amount of pollutant per cell. For exampleStauber et al. (2002) found that as the initial algalcell density of the fresh-water algae Chlorella sp.and Selenastrum capricornutum was increased, thetoxicity of copper decreased. The initial cell densi-ty has been proved to be an important factor which

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modulates results in bioassays. To use a low cellnumber might be more similar to natural habitatconditions, but using very few cells, in laboratoryexperimental conditions, may introduce some er-rors in endpoint values. One reason is that cell via-bility might be damaged prior to the heavy metalexposure (for example, because of cell manipula-tion) and that might accelerate cell death, indepen-dently of the real toxic effect of a heavy metal.

g) Physiological stage of the micro-organism.Another important point to be considered in eco-toxicological studies is the physiological stage ofthe reporter cell. It has been pointed out by Nils-son (1989) that Tetrahymena cells in stationaryphase are more sensitive to Cd than those in the ex-ponential growth phase at the same Cd concentra-tion because the increased acidity liberates more ofthe toxic form of Cd.

h) Bio-geographical source or the “strain ef-fect”. Besides the above-mentioned factors, whichintroduce a high variability in the experimentalprocedure of ciliate ecotoxicological bioassays,and make it difficult to establish comparisons, an-other important point to be considered in ciliateecotoxicology might be named the “strain effect”.Strains or isolates of the same ciliate species fromdifferent bio-geographical origin or habitat canshow different heavy metal resistance levels. Forinstance, an ecotoxicological study (unpublishedwork) carried out with different strains of Colpodasteinii (the most frequent and representative soilciliate) have shown very different LC50 (Cd, Zn orCu) values, in four different strains isolated fromhabitats polluted or not polluted with heavy met-als.

After considering all these sources of variabilityin ciliate heavy metal bioassays, we propose thatpeople working in ciliate ecotoxicological studiesshould agree to standardise and validate standardciliate-specific bioassays, as have been achieved inother organisms (microalgae, Daphnia, etc).

Features that make ciliates suitable aswhole-cell or molecular biosensors forheavy metal monitoring

1. Heavy metal bioaccumulation and the useof specific heavy metal fluorophores

Bioaccumulation is the most common heavymetal resistance mechanism among ciliates

(Martín-González et al. 1999). Such metallic bioac-cumulation can be revealed by fluorescence mi-croscopy. In 2001, we reported for the first time inciliates, (unpublished work) the use of specificheavy metal fluorophores to distinguish ciliates ex-posed to sub-lethal metallic concentrations fromcontrols. Two specific fluorophores have been ap-plied to diverse ciliates after Zn or Cd treatment;TSQ [N-(6-metaoxy-8-quinolyl-p-toluenesulfon-amide)] (Molecular Probes) which is selective forZn2+ in the presence of physiological concentra-tions of Ca2+ and Mg2+ ions, and bis-BTC (tetra-ammonium salt) (Molecular Probes) which bindsCd2+/Cd0. An example of TSQ application isshown in Fig. 1. Results from fluorescence mi-croscopy suggest that this method is only useful tolocate cytoplasmic metallic deposits when cells areexposed to high heavy metal concentrations. Fur-thermore, some fluorophores (for instance, TSQ)seem to be more specific and sensitive than others.But, in any case, clear differences between treatedcells and controls (Fig. 1) reveal that this method-ology might be useful in heavy metal biomonitor-ing. The fluorescence of cell populations treatedwith heavy metals might be measured by flow cy-tometry, so obtaining a quantification of heavymetal bioaccumulation at cell population level.This methodology might be a useful tool to detectheavy metals in urban wastewater treatment plantsusing ciliates as whole-cell biosensors.

2. Fluorescent detection of ROS (ReactiveOxygen Species) generated in ciliates by heavymetals

Heavy metals may induce (directly or indirectly)oxidative stress in both prokaryotic and eukaryoticcells. Heavy metals with redox activity (Cu, amongothers) can directly give rise to ROS byFenton/Haber-Weiss reactions or auto-oxidation.Besides, heavy metals without redox activity (likeCd or Zn) can also indirectly generate oxidativestress by blocking or decreasing cellular antioxi-dant defences. These defences may be enzymatic(antioxidant enzymes, such as; glutathione peroxi-dase, catalase or superoxide dismutase) or non-en-zymatic (glutathione and metallothioneins, whichhave a protective effect against oxidative stress).

The formation of hydrogen peroxide (H2O2)and superoxide anions as a result of the action ofheavy metals has been analysed in several ciliatesby using three different fluorophores (2′,7′-dichlo-

464 J. C. Gutièrrez et al.

Besides, it assists in the glutathione peroxidase re-action, which is involved in the detoxification ofhydroperoxide (ROOH).

Treatment with BSO (buthionine sulfoximine),which is an inhibitor of γ-glutamylcysteine syn-thetase (the first enzyme involved in GSH biosyn-thesis), leads to decreased cellular GSH levels, andits application can provide a useful experimentalmodel of GSH deficiency (Anderson 1998). Usingthis inhibitor, we have carried out several experi-ments (unpublished data); intracellular moleculeswith thiol groups (GSH, metallothioneins andothers) were measured by flow cytometry, usingthe fluorophore monobromobimane (mBBr)(Molecular Probes), on cell populations previously

rofluorescein diacetate, dihydrorhodamine 123and dihydroethidium) (unpublished work). Bothfluorescence microscopy with quantitative imageanalysis on fixed cells (Fig. 2) and flow cytometryusing living cell populations, have been shown tobe useful tools to distinguish between controls andcells treated with heavy metals.

Glutathione (GSH) is a tripeptide (γ-Glu-Cys-Gly) which is found in eukaryotic and prokaryoticcells, and it is the most abundant intracellular thiol.This molecule has important cellular functions, forinstance; protection against oxidative stress, be-cause it may react non-enzymatically with ROS,and it may also react with heavy metals (like phy-tochelatins) by its thiol groups (Anderson 1998).

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Fig. 1. TSQ application in cells of Colpoda steinii AZ1. (A): Putative Zn deposits (cytoplasm regions with an in-tense red fluorescence) (e.g. at arrow) in a cell treated for 24 h with 50 mg l–1 of Zn in C0.25E1 medium (Martín-González et al. 1991). (B): Control (without heavy metal treatment ).

Fig. 2. Application of dichlorofluorescein diacetate to reveal intracellular H2O2 formation (oxidative stress) byheavy metal exposure in Tetrahymena sp. RT2. (A): Cell treated for 1 h with 2 mg l–1 of Cu in C0.25E1 medium(Martín-González et al. 1991). (B): Control (without heavy metal treatment).

treated or not with BSO and heavy metals (Cu orCd). These experiments, performed on the ciliateTetrahymena sp. (strain RT1), demonstrated theimportance of GSH in cellular protection fromheavy metals. Results revealed that in cells treatedwith BSO the LC50 (Cu) value decreased (1.8 mgl–1) compared with controls (2.4 mg l–1), indicatingincreased Cu toxicity in the absence of GSH. Inthe presence of Cu or Cd (at sub-lethal concentra-tions) an induction of biosynthesis of moleculeswith thiol groups was detected, while in presenceof BSO (the inhibitor of GSH biosynthesis) theamount of these thiol molecules decreases. This in-dicated that GSH has an important role in cellularprotection against heavy metals, and, likewise, itmight be a useful molecular biomarker for moni-toring cell populations exposed to heavy metals.

3. Ciliate metallothioneins:candidates for use as molecular biosensors

Ciliate Cd-metallothioneins (Cd-MTs) presentunique features in comparison with the usual MTsfrom other organisms (Piccinni et al., 1999; Shanget al., 2002). The main differences between themare: (1) ciliate Cd-MTs have higher molecular mass-es (11–17 kDa) than standard MTs (<7–10 kDa); (2)they are unusually rich in cysteine (Cys, the aminoacid involved in the metal chelating capacity of theprotein) (31–48 Cys residues/molecule), in con-trast to standard MTs (18–23 Cys residues/molecule), and among Cys motifs, the CCC motifis found (so far) in all Cd-MT isoforms of ciliatesand only one Cd-MT from the common brandlingworm Eisenia fetida; (3) aromatic amino acids arenot present in standard MTs, however they appearin two ciliate Cd-MT isoforms (unpublishedwork). Furthermore, ciliate Cd-MTs are over-ex-pressed within a few minutes under heavy metal ex-posure. In mammalian MTs all cysteine residues areknown to participate in the coordination of 7 molof Cd or Zn per mol of MT, thereby satisfying theCd7 (Cys)20 stoichiometry for Cd-MTs. If ciliateCd-MTs also satisfy this stoichiometry and all Cysresidues are used in the metal-binding process, thetheoretical Cd-binding capacity of ciliate Cd-MTsis considerably higher than that of mammalianMTs. At least two ciliate Cd-MT isoforms are evo-lutionarily highly conserved among ciliates, be-cause we have found very similar sequences in dis-tantly related ciliates (unpublished work).

All these properties make it possible that theseproteins can be very good candidates to be used as

molecular biomarkers or as the biological element(bioreceptor) in the design of molecular biosen-sors. MTs are stress proteins and show the threefeatures that any stress protein needs to possess tobe considered as a biomarker of pollution(Bierkens 2000): (a) they are part of the cellularprotective response; (b) their synthesis is likely tobe induced by a large number of chemical or phys-ical factors; and (c) they are highly conserved in allorganisms. So, MTs have been considered as excel-lent biomarkers for heavy metal and other envi-ronmental pollutant monitoring (Dallinger et al.2000). Recently, a fragment of MT has been cova-lently immobilized onto piezoelectric crystals tostudy the complexation of MT with Cd and Znions (Saber and Piskin 2002); this might a prelimi-nary step in the design of a future molecularbiosensor.

Ciliate Cd-MTs can offer us many interestingpossibilities for the detection of heavy metals inpolluted environmental samples, for instance, thepromoter of one Tetrahymena thermophila Cd-MT isoform has proved to be a robust inducible-repressible promoter which facilitates gene knock-outs, conditional expression and over-expressionof both homologous and heterologous genes(Shang et al. 2002). This promoter might be used in“turn on “ assays using whole-cell biosensors, afterfusion to a quantifiable molecular reporter (likegreen fluorescent protein). Ciliate Cd-MT frag-ments might be used as bioreceptors in the designof classical biosensors, after studying the complex-ation of the immobilized oligopeptide with heavymetal ions. Likewise, gene fragments of ciliate Cd-MTs could be used in the construction of macro-or micro-arrays, so the fast expression of thesegenes under heavy metal exposure might in the fu-ture provide a good molecular tool to detect thepresence of heavy metals in polluted environmen-tal samples.

Acknowledgements: The research on ciliate-heavymetal interactions, ciliate metallothioneins and ROSformation is supported by grant projects I+D(BOS2002-01067) and 07M/0029/2002 (CAM).

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