• E-mail
• Print
• Comment
• Font Size
• Digg
• del.icio.us
• Discuss article

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

Posted on: Sunday, 14 March 2004, 06:00 CST

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

Keywords: Biomarkers; Biosensors; Ciliates; Fluorophores; Heavy metals; Metallothioneins.

Introduction: General concepts

The term biomarker was originally defined as "a detectable biochemical or physiological alteration or cellular manifestation brought about by environmental stress". In the present context, the concept of a biomarker is used in a more restrictive sense, namely "a sub-lethal biochemical change resulting from individual exposure to a toxic pollutant" (Gadd 1992). Therefore, any cellular system which undergoes a detectable physiological alteration under the influence of a specific environmental stress or pollutant can be considered as a cell biomarker, and molecules involved in this alteration or physiological change may be considered molecular biomarkers (e.g. metallothionein biosynthesis under heavy metal exposure).

On the other hand, the classical biosensor concept involves the existence of two components; a bioreceptor (biological material) and a physicochemical transducer. The bioreceptor might be a biomolecule or a whole cell that recognises the target (heavy metal), whereas the transducer converts the recognition event into a measurable signal. The sensing elements (bioreceptors) might be enzymes, antibodies, DNA molecules, cell receptors and organelles, or whole cells of micro-organisms as well as of animals or plants, and the transducer may be electrochemical or mechanical, optical or acoustic. The function of a biosensor depends on the biochemical specificity of the biologically active material. The choice of the biological material will depend on a number of factors, including the nature of the pollutant to be detected.

Recently, the concept of the whole-cell biosensor has been introduced by several authors (D'Souza 2001; Belkin 2003), as a very useful alternative to classical biosensors. A whole-cell biosensor uses the whole prokaryotic or eukaryotic cell as a single reporter incorporating both bioreceptor and transducer elements. In general, living systems to be used as whole-cell biosensors are experimentally modified to incorporate the transducer capacity.

In environmental biomonitoring, global parameters such as bioavailability, toxicity and genotoxicity can not be tested using molecular recognition or chemical analysis, but can only be assayed using whole cells. Obviously, in these bioassays the question to be resolved is not "What toxicants does the sample contain?", but rather "How toxic is the sample?"

Two types of bioassays using whole-cell biosensors may be considered: "turn off" and "turn on" assays. In "turn off" assays (which are similar to general microbial toxicological bioassays), the sample toxicity is estimated from the degree of inhibition of a cellular activity that is a normally continuous (e.g. inhibition of growth, respiration or metabolism, motility or the biosynthesis of a specific molecule), and is based on the measurement of a decrease in growth rate, light (fluorescence/bioluminescence) emission, colour- less cell population, motility, etc. as a function of sample toxic concentration. For instance, a good example of a "turn off" assay using ciliates, may be a reported rapid bioassay to detect mycotoxins using a melanin precursor overproducer mutant of the ciliate Tetrahymena thermophila (Martin-Gonzalez et al. 1997). On the other hand, m "turn on" assays a quantifiable molecular reporter is fused to a specific gene promoter (like a nietallothionein promoter), known to be activated by the target chemical or environmental pollutant (such as a heavy metal).

Advantages in using micro-organisms as whole-cell biosensors/ biomarkers in environmental pollution monitoring

As indicated above, biological systems, particularly micro- organisms, provide the best ways to assess pollutant bioavailability. This ecotoxicological parameter is really important in heavy metal pollution because metal ions can bind to abiotic surfaces and ligands (like particulate organic matter, clay and metal (hydroxides/oxides), which may strongly influence their bioavailability and, therefore, their cellular toxic effects. For this reason the organic matter content and other factors (like pH) of the culture medium are important parameters to be considered in any heavy metal toxicological bioassay. Furthermore, the use of micro-organisms provides data on the biological effects following exposure to heavy metals, which may be extended to other more complex living systems. Also, they can provide data on heavy metal interactions (synergistic or antagonistic toxic effects) in metal mixtures. Heavy metal pollution is usually multiple (two or more different metals are typically present), and the cellular toxic effect of a metal can vary in response to the presence of other heavy metals. For instance, calcium and zinc have an antagonistic effect, like cadmium and zinc, whereas mercury increases the toxicity to zinc (Nies 1999). We have observed in the soil ciliate Colpoda steinii that the medium lethal concentration (LC^sub 50^) of cadmium increases about 15 times in the presence of zinc at sub- lethal concentrations (unpublished work).

In general, micro-organisms show a rapid growth rate, reaching a large cell population in a short time, which is important in some ecotoxicological assays to obtain an amplified response for detecting the pollutant-induced change, when micro-organisms are used as whole-cell biomarkers or biosensors. Besides, it is possible to use genetically engineered micro-organisms, which are very useful in "turn on" assays, and to make up appropriate genetic constructions to detect a specific pollutant (e.g. heavy metal- specific biosensors). Likewise, specific mutants or micro-organisms with several gene reporters might be used. Genotoxicity studies are simpler and more practicable using micro-organisms because they can more easily be subjected to genetic dissection.

Additional advantages of using ciliates as whole-cell biomarkers/ biosensors

Ciliates are eukaryotic micro-organisms; they have all the advantages previously reported, and they present at least two additional advantages:

a) In contrast to bacteria and yeasts, ciliates are unicellular organisms without a cell wall in the vegetative stage. A major limitation to the use of bacteria or yeasts as whole-cell biosensors is the uncertainty concerning diffusion of substrates and products through the cell wall, which may result in a slower or less effective response as compared to enzyme-based sensors. To obviate this problem such cells must be permeabilised using physico- chemical or enzymatic methods to allow the free diffusion of small molecules across the cell membrane, but the cells are no longer normal viable organisms. The use of ciliates, instead of bacteria or yeasts, might avoid this serious problem. The absence of a cell wall in these eukaryotic micro-organisms might offer a higher sensitivity to environmental pollutants, and, therefore, a faster cellular response.

b) They are eukaryotic micro-organisms with some metabolic traits that are more similar to those of human cells than are bacteria or yeasts. Pilot genome projects in two model ciliates often used in ecotoxicological studies, Tetrahymena and Paramecium (Dessen et al. 2001; Turkewitz et al. 2002), have shown that they share a higher degree of functional conservation with human genes than do other microbial model eukaryotic micro-organisms. This is shown by better matches of relevant ciliate coding sequences to those in humans, as compared with non-ciliate microbial models. Therefore, it seems to be more reasonable to use these eukaryotic cells in ecotoxicological studies as models for humans, and represents an alternative to animal tests.

Diversity of experimental conditions and methodological approaches in ciliate heavy metal bioassays

To select ciliates in order to design whole-cell biosensors for heavy metal environmental monitoring, we should first determine basic ecotoxicological parameters (e.g. resistance/tolerance or sensitivity to a specific metal) for that ciliate strain under controlled laboratory conditions. After reviewing published data on heavy metal ecotoxicological analysis using ciliates, we conclude that the diversity of experimental conditions and methodological approaches is generally too great to make comparisons between the results using different ciliates. The following points illustrate this generalized and serious problem:

a) Heavy metal concentration parameters. Metal concentrations are expressed in diverse forms, generally as weight/volume (ppm or mg/ 1) or molarity (M or mM). If authors report experimental data properly, \the conversions among these are easily possible, but problems arise if concentrations are expressed in less conventional ways, as a fraction of total proteins or of dry weight, for example.

b) Culture medium composition. This can represent a significant problem in some circumstances, because the concentrations of organic matter and dissolved salts or the presence of sediments or clay are not usually reported. These parameters, as well as some pH changes, reduce the heavy metal availability and, therefore, decrease the toxic impact. For instance, in Tetrahymena pyriformis growth is not affected up to a dose of 8 mg l^sup -1^ Cd (in 2% proteose-peptone and 0.1% yeast extract medium) (Sauvant et al. 1999). By contrast, Schaefer et al. (1994) reported an EC50 at 48 h, of 0.78 mg I^sup - 1^ Cd (in a medium composed by 1% proteose-peptone and 1% yeast extract) for the same ciliate. A protective effect of organic matter has also been observed in other ciliates (MartinGonzalez et al. 1999).

c) Assessment of toxic effect. In general, ecotoxicological assays using ciliates have been conducted to assess sub-lethal effects, such as inhibition of growth, variation in feeding rate, inhibition of respiration, motility, etc.

d) Endpoints. The LD^sub 50^/LC^sub 50^ (median lethal dose/ median lethal concentration) is the most frequently used toxicological endpomt in ciliates, but other measures of physiological effect are also used, such as the IG50 (median growth inhibition concentration), also named the EC50 (effective concentration for 50% growth inhibition) or IC50 (median inhibitory concentration), and other more unusual forms, like LOED/LOEC (lowest observed effective dose or concentration) and TLm (median tolerance limit).

e) Duration of heavy metal exposure. In ciliate toxicological studies the duration of heavy metal exposure used in assays is also very diverse. The majority of authors measure the toxic effect at 24 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 diverse parameter among ciliate ecotoxicological assays. The range of cell concentrations is really large; some authors use concentrated cell cultures (e.g. about 10^sup 3^ cells ml^sup -1^; Martin-Gonzalez et al. 1997) and others very few cells (e.g. about 12 cells; Madoni et al. 1994). Obviously, LC^sub 50^ is lower in cultures with low cell number, because of the increase in amount of pollutant per cell. For example Stauber et al. (2002) found that as the initial algal cell density of the fresh-water algae Chlorella sp. and Selenastrum capricornutum was increased, the toxicity of copper decreased. The initial cell density has been proved to be an important factor which modulates results in bioassays. To use a low cell number might be more similar to natural habitat conditions, but using very few cells, in laboratory experimental conditions, may introduce some errors in endpoint values. One reason is that cell viability might be damaged prior to the heavy metal exposure (for example, because of cell manipulation) and that might accelerate cell death, independently of the real toxic effect of a heavy metal.

g) Physiological stage of the micro-organism. Another important point to be considered in ecotoxicological studies is the physiological stage of the reporter cell. It has been pointed out by Nilsson (1989) that Tetrahymena cells in stationary phase are more sensitive to Cd than those in the exponential growth phase at the same Cd concentration because the increased acidity liberates more of the toxic form of Cd.

h) Bio-geographical source or the "strain effect". Besides the above-mentioned factors, which introduce a high variability in the experimental procedure of ciliate ecotoxicological bioassays, and make it difficult to establish comparisons, another important point to be considered in ciliate ecotoxicology might be named the "strain effect". Strains or isolates of the same ciliate species from different bio-geographical origin or habitat can show different heavy metal resistance levels. For instance, an ecotoxicological study (unpublished work) carried out with different strains of Colpoda stemii (the most frequent and representative soil ciliate) have shown very different LC^sub 50^ (Cd, Zn or Cu) values, in four different strains isolated from habitats polluted or not polluted with heavy metals.

After considering all these sources of variability in ciliate heavy metal bioassays, we propose that people working in ciliate ecotoxicological studies should agree to standardise and validate standard ciliate-specific bioassays, as have been achieved in other organisms (microalgae, Daphnia, etc).

Features that make ciliates suitable as whole-cell or molecular biosensors for heavy metal monitoring

1. Heavy metal bioaccumulation and the use of specific heavy metal fluorophores

Bioaccumulation is the most common heavy metal resistance mechanism among ciliates (Martin-Gonzalez et al. 1999). Such metallic bioaccumulation can be revealed by fluorescence microscopy. In 2001, we reported for the first time in ciliates, (unpublished work) the use of specific heavy metal fluorophores to distinguish ciliates exposed to sub-lethal metallic concentrations from controls. Two specific fluorophores have been applied to diverse ciliates after Zn or Cd treatment; TSQ [N-(6-metaoxy-8-quinolyl-p- toluenesulfonamide)] (Molecular Probes) which is selective for Zn^sup 2+^ in the presence of physiological concentrations of Ca^sup 2+^ and Mg^sup 2+^ ions, and bis-BTC (tetraammonium salt) (Molecular Probes) "which binds Cd^sup 2+^/Cd^sup 0^. An example of TSQ application is shown in Fig. 1. Results from fluorescence microscopy suggest that this method is only useful to locate cytoplasmic metallic deposits when cells are exposed to high heavy metal concentrations. Furthermore, some fluorophores (for instance, TSQ) seem to be more specific and sensitive than others. But, in any case, clear differences between treated cells and controls (Fig. 1) reveal that this methodology might be useful in heavy metal biomonitoring. The fluorescence of cell populations treated with heavy metals might be measured by flow cytometry, so obtaining a quantification of heavy metal bioaccumulation at cell population level. This methodology might be a useful tool to detect heavy metals in urban wastewater treatment plants using ciliates as whole- cell biosensors.

2. Fluorescent detection of ROS (Reactive Oxygen Species) generated in ciliates by heavy metals

Heavy metals may induce (directly or indirectly) oxidative stress in both prokaryotic and eukaryotic cells. Heavy metals with redox activity (Cu, among others) can directly give rise to ROS by Fenton/ Haber-Weiss reactions or auto-oxidation. Besides, heavy metals without redox activity (like Cd or Zn) can also indirectly generate oxidative stress by blocking or decreasing cellular antioxidant defences. These defences may be enzymatic (antioxidant enzymes, such as; glutathione peroxidase, catalase or Superoxide dismutase) or non- enzymatic (glutathione and metallothionems, which have a protective effect against oxidative stress).

The formation of hydrogen peroxide (H^sub 2^O^sup 2^) and Superoxide anions as a result of the action of heavy metals has been analysed in several ciliates by using three different fluorophores (2',7'-dichlorofluorescein diacetate, dihydrorhodamine 123 and dihydrocthidium) (unpublished work). Both fluorescence microscopy with quantitative image analysis on fixed cells (Fig. 2) and flow cytometry using living cell populations, have been shown to be useful tools to distinguish between controls and cells treated with heavy metals.

Glutathione (GSH) is a tripeptide ([gamma]-Glu-CysGIy) which is found in eukaryotic and prokaryotic cells, and it is the most abundant intracellular thiol. This molecule has important cellular functions, for instance; protection against oxidative stress, because it may react non-enzymatically with ROS, and it may also react with heavy metals (like phytochelatins) by its thiol groups (Anderson 1998). Besides, it assists in the glutathione peroxidase reaction, which is involved in the detoxification of hydroperoxide (ROOH).

Fig. 1. TSQ application in cells of CoIpoda steinii AZl. (A): Putative Zn deposits (cytoplasm regions with an intense red fluorescence) (e.g. at arrow) in a cell treated for 24 h with 50 mg I^sup -1^ of Zn in C0.25E1 medium (Martin-Gonzalez et al. 1991). (B): Control (without heavy metal treatment ).

Fig. 2. Application of dichlorofluorescein diacctatc to reveal intracellular H^sub 2^O^sub 2^ formation (oxidativc stress) by heavy metal exposure in Tetrahymena sp. RT2. (A): Cell treated for l h with 2 mg l^sub -1^ of Cu in CO.25El medium (Martin-Gonzalez et al. 1991). (B): Control (without heavy metal treatment).

Treatment with BSO (buthiomne sulfoximme), which is an inhibitor of [gamma]-glutamylcysteine synthetase (the first enzyme involved in GSH biosynthesis), leads to decreased cellular GSH levels, and its application can provide a useful experimental model of GSH deficiency (Anderson 1998). Using this inhibitor, we have carried out several experiments (unpublished data); intracellular molecules with thiol groups (GSH, metallothioneins and others) were measured by flow cytometry, using the fluorophore monobromobimane (mBBr) (Molecular Probes), on cell populations previously treated or not with BSO and heavy metals (Cu or Cd). These experiments, performed on the ciliate Tetrahymena sp. (strain RTl), demonstrated the importance of GSH in cellular protection from heavy metals. Results revealed that in cells treated with BSO the LC^sub 50^ (Cu) value decreased (1.8 mg H) compared with controls (2.4 mg l^sup -1^), indicating increased Cu toxicity in the absence of GSH. In the presence of Cu or Cd (at sub-lethal concentrations) aninduction of biosynthesis of molecules with thiol groups was detected, while in presence of BSO (the inhibitor of GSH biosynthesis) the amount of these thiol molecules decreases. This indicated that GSH has an important role in cellular protection against heavy metals, and, likewise, it might be a useful molecular biomarker for monitoring cell populations exposed to heavy metals.

3. Ciliate metallothioneins: candidates for use as molecular biosensors

Ciliate Cd-metallothioneins (Cd-MTs) present unique features in comparison with the usual MTs from other organisms (Piccinni et al., 1999; Shang et al., 2002). The main differences between them are: (1) ciliate Cd-MTs have higher molecular masses (11-17 kDa) than standard MTs (<7-10 kDa); (2) they are unusually rich in cysteine (Cys, the amino acid involved in the metal chelating capacity of the protein) (31-48 Cys residues/molecule), in contrast to standard MTs (18-23 Cys residues/ molecule), and among Cys motifs, the CCC motif is found (so far) in all Cd-MT isoforms of ciliates and only one Cd- MT from the common brandling worm Eisenia fetida; (3) aromatic amino acids are not present in standard MTs, however they appear in two ciliate Cd-MT isoforms (unpublished work). Furthermore, ciliate Cd- MTs are over-expressed within a few minutes under heavy metal exposure. In mammalian MTs all cysteine residues are known to participate in the coordination of 7 mol of Cd or Zn per mol of MT, thereby satisfying the Cd^sub 7^ (Cys)^sub 20^ stoichiometry for Cd- MTs. If ciliate Cd-MTs also satisfy this stoichiometry and all Cys residues are used in the metal-binding process, the theoretical Cd- bmding capacity of ciliate Cd-MTs is considerably higher than that of mammalian MTs. At least two ciliate Cd-MT isoforms are evolutionarily highly conserved among ciliates, because we have found very similar sequences in distantly related ciliates (unpublished work).

All these properties make it possible that these proteins can be very good candidates to be used as molecular biomarkers or as the biological element (bioreceptor) in the design of molecular biosensors. MTs are stress proteins and show the three features that any stress protein needs to possess to be considered as a biomarker of pollution (Bierkens 2000): (a) they are part of the cellular protective response; (b) their synthesis is likely to be induced by a large number of chemical or physical factors; and (c) they are highly conserved in all organisms. So, MTs have been considered as excellent biomarkers for heavy metal and other environmental pollutant monitoring (Dallinger et al. 2000). Recently, a fragment of MT has been covalcntly immobilized onto piezoelectric crystals to study the complexation of MT with Cd and Zn ions (Saber and Piskm 2002); this might a preliminary step in the design of a future molecular biosensor.

Cihate Cd-MTs can offer us many interesting possibilities for the detection of heavy metals in polluted environmental samples, for instance, the promoter of one Tetrahymena thermophila CdMT isoform has proved to be a robust induciblerepressible promoter which facilitates gene knockouts, conditional expression and over- expression of both homologous and heterologous genes (Shang et al. 2002). This promoter might be used in "turn on " assays using whole- cell biosensors, after fusion to a quantifiable molecular reporter (like green fluorescent protein). Ciliate Cd-MT fragments might be used as bioreceptors in the design of classical biosensors, after studying the complexation of the immobilized oligopeptide with heavy metal ions. Likewise, gene fragments of ciliate CdMTs could be used in the construction of macroor micro-arrays, so the fast expression of these genes under heavy metal exposure might in the future provide a good molecular tool to detect the presence of heavy metals in polluted environmental samples.

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

References

Anderson M. E. (1998): Glutathione: an overview of biosynthesis and modulation. Chem.-Biol. Interac. 111-112, 1-14.

Belkin S, (2003): Microbial whole-cell sensing systems of environmental pollutants. Curr. Opin. Microbiol. 6, 206-212.

Bierkens J. G. E. A. (2000): Applications and pitfalls of stress- proteins in biomonitoring. Toxicology 153, 61-72.

Dallinger R., Berger B., Gruber C., Hunziker P. and Sturzenbaum S. (2000): Metallothioneins in terrestrial invertebrates: structural aspects, biological significance and implications for their use as biomarkers. Cell. MoI. Biol. 46, 331-346.

Dessen P., Zaguiski M., Gromadka R., Plattner H., Kissmehl R., Meyer E., Betermicr M., Schultz J. E., Linder J. U., Pcarlman R. E., Kung C., Forney J., Saur B. H., Van Horten J. E., Keller A-M., Froissad M., Sperling E. and Cohen J. (2001): Paramecium genome survey: a pilot project. Trends Genet. 17, 306-308.

D'Souza S. F. (2003): Microbial biosensors. Biosensors Bioelectron. 16, 337-353.

Gadd G. M. (1992): Heavy metal pollutants: Environmental and Biotechnological aspects. In: Eederberg J. (ed.): Encyclopedia of Microbiology, pp. 351-360. Academic Press, Eondon and New York.

Madoni P., Davoli D. and Gorbi G. (1994): Acute toxicity of lead, chromium and other heavy metals to ciliatcs from activated sludge plants. Bull. Environ. Contam. Toxicol. 53, 420-425.

Martin-Gonzalez A., Benitez E., Soto T., Rodriguez de Eecea J. and Gutierrez J. C. (1997): A rapid bioassay to detect mycotoxins using a melanin precursor overproducer mutant of the ciliate Tetrahymena thermophila. Cell Biol. Intern. 21, 213-216.

Martin-Gonzalez A., Benitez E., Cortadellas N. and Gutierrez J. C. (1991): Detection of antigenic cyst wall elements in Colpoda inflata: an immunoelectron microscopic study and immunoblotting identification of cyst wall polypeptides. Cell Molec. Biol. 37, 21- 27.

Martin-Gonzalez A., Diaz S., Jareno C. and Gutierrez J. C. (1999): The use of protists in ecotoxicology: applications and perspectives. In: Pandalai S.G. (ed.): Recent research developments in microbiology, pp. 93-111. Research Signpost, India.

Nies D. H. (1999): Microbial heavy-metal resistance. Appl, Microbiol. Biotechnol. 51, 730-750.

Nilsson J. R. (1989): Tetrahymena in cytotoxicology: with special reference to effects of heavy metals and selected drugs. Europ. J. Protistol. 25, 2-25.

Piccinni E., Bertaggia D., Santovito G., Miceli C. and Kracu A. (1999): Cadmium metallothionein gene of Tetrahymena pyriformis. Gene. 234, 51-59.

Saber R. and Piskin E. (2002): Investigation of complexation of immobilized metallothionein with Zn (II) and Cd (II) ions using piezoelectric crystals. Biosensors Bioelectron. 18,1-8.

Sauvant M. P., Pepin D. and Piccinni E. (1999): Tetrahymena. pyriformis: a tool for toxicological studies. A review. Chemosphere 38, 1631-1669.

Schacfer H., Hettler H., Pritsche U., Pitzen G., Roederer G. and Wenzel A. (1994): Biotest using unicellular algae and cihates for predicting long-term effects of toxicants. Ecotoxicol. Environ. Saf. 27, 64-81.

Shang Y., Song X., Bowen J., Corstanje R., Gao Y., Gacrtig J. and Gorovsky M. A. (2002): A robust inducible-rcprcsible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophiia. Proc. Natl. Acad. Sei. USA. 99, 3734-3739.

Stauber J. L., Franklin M. N. and Adams M. S. (2002): Applications of flow cytometry to ecotoxicology testing using microalgae. Trends Biotechnol. 20, 141-143.

Turkewitz A., Orias E. and Kapler G. (2002): Functional genomics: the coming of age for Tetrahymena thermophiia. Trends Genet. 18, 35- 40.

Juan Carlos Gutierrez*, Ana Martin-Gonzalez, Silvia Diaz and Ruth Ortega

Departamento de Microbiologia-lll, Facultad de Biologia, C/. Jose 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

* corresponding author

Copyright Urban & Fischer Verlag Dec 2003

More News in this Category