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Similar topics of scientific paper in Environmental engineering , author of scholarly article — S. J. Hoeger, D. Schmid, J. F. Blom, B. Ernst, D. R. Dietrich


Academic research paper on topic " Analytical và Functional Characterization of Microcystins MC-RR & MC-RR: Consequences for Risk Assessment? "


Environ. Sci. Technol. 2007, 41, 2609-2616

Analytical & Functional Characterization of Microcystins MC-RR and

MC-RR: Consequences for Risk Assessment?

S. J. HOEGER.t D. SCHMID,t § J. F. BLOM,* B. ERNST,+ AND D. R. DIETRICH*-1

Environmental Toxicology, University of Konstanz, Germany, và Limnological Station, Institute of Plant Biology, University of Zürich, Switzerland

The microcystin (MC) producing P.. rubescens occurs in pre-alpine lakes and may impact fishery success, bathing, & raw water unique. P. rubescens extracts, characterized via LC-MS, contained the two MC-RR variants MC-RR and MC-RR. The protein-phosphatase-inhibition assay (cPPIA with phosphatases 1 and 2A) in its capability lớn quantify MC-RR, MC-RR, and MC-RR was compared lớn HPLC-DAD and anti-Adda-ELISA. The IC50 values (PP1 and PP2A) determined for MC-LR, MC-RR, và MC-RR were in the same range (1.93.8 và 0.45-0.75 nM). A 50-fold higher concentration of MC-RR (29.8 nM) was necessary to lớn inhibit the PP2A by 50%. The PPHC50 of MC-RR was 22fold higher (56.4 nM) than those of the other MCs, suggesting that specific structural characteristics are responsible for its weaker PPI capathành phố. Western blots demonstrated that MC-RR does not covalently bind to lớn PP1. MC-RR has comparable in vivo LD50 values lớn MC-RR, despite a far lower PP-inhibiting capathành phố, suggesting that toxicodynamic and toxicokinetic characteristics of MC-RR are responsible for its high in vivo toxiđô thị. The data demonstrate that cPPIA analysis of MC-RR-containing samples prevent reliable MC determination & lead to lớn underestimation of potential toxicity.

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Introduction

Water blooms containing toxic cyanobacteria occur ubiquitously worldwide. These blooms may represent a hazard khổng lồ humans và to the environment (1). A cyanobacterial genus well-known for its potential to produce toxins is the filamentous Planktothrix, frequently foundin European lakes (2, 3). In Lake Ammersee (Southern Germany), P. rubescens is one of the dominant planktonic species, và it regularly attains high densities & produces several microcystin congeners (MCs) (2, 4, 5). The principal structure of the approximately 80 different MCs is cyclo-(D-Ala1-L-X2-D-

uni-konstanz.de.

t University of Konstanz.

*University of ZUrich.

§ Current address: Department of Bioorganic Chemistry, Max Planchồng Institute for Chemical Ecology, Jena, Germany.

MAsp3-L-Z4-Adda5-D-Glu6-MDha7), whereby MAsp3 stands for erythro-^-methylaspartic acid, Addomain authority for 4£,6£-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, và MDha7 for N-methyldehydroalanine. X & Z represent two variable amino acids with 15 & 12 variations reported, respectively. Structural modifications are reported for the remaining amino acids; however, demethylation on MAsp, MDha, or Addomain authority and/or esterification of glutamic acid and Z-configuration of Addomain authority occur most frequently. Nodularins (NODs), toxic cyanobacterial pentapeptides with a similar principal structure, i.e., cyclo-(D-MAsp1-L-Arg2-Adda3-D-Glu4-MDhb5), whereby MDhb5 represents N-methyldehy-drobutyric acid, are produced by the brackish water cyano-bacterium Nodularia spumigena (6). Although the functional và ecological role(s) of MCs and NODs is unknown (7), it is beyond dispute that MCs & NODs are responsible for adverse health effects in humans & animals (8).

Following ingestion MCs & NODs are absorbed from the gastro-intestinal tract, reach the liver via the portal vein, và are actively taken up from the blood inlớn hepatocytes via bile acid transporters (9). MCs và NODs are specific và highly effective inhibitors of the catalytic subunits of several serine/threonine protein-phosphatases (PP) (10). As a consequence of PP-inhibition, cellular phospho-proteins (e.g., micro- và intermediate-filaments, tau proteins, tumor suppressors, etc.) are hyperphosphorylated. Following acute intoxication, cytoskeletal protein hyperphosphorylation leads lớn disintegration of the cytoskeletal scaffold with subsequent apoptosis, necrosis, và finally, liver failure (11). In contrast, chronic exposure results in hepatocyte proliferation, most likely due to lớn hyperphosphorylation of tumor suppressor proteins và, finally, in liver tumor promotion (reviewed in ref 8). NODs may have tumor-initiating as well as promoting activity (12). Consequently, the International Agency for Cancer Research (IARC) classified MC-LR in the 2B category, probably carcinogenic khổng lồ humans (13).

Studies aimed at defining binding of MCs to PP1 và PP2A (14, 15) reported that the MDha residue of MC-LR covalently binds to the cysteine-273 and cysteine-266 residues of PP1 and PP2A, respectively, although binding does not induce changes in MC-LR conformation (16). This was corroborated via crystallographic structure-analysis of the MC-LR-PP1 complex (17). In contrast, NOD does not covalently bind khổng lồ protein-phosphatases (15,18). Moreover, the >10 A distance between the ^-carbon of the Mdhb residue & the sulfur atom of the Cys-273 side chain of the PP1-catalytic-subunit (18) most likely renders NOD incapable of covalent binding to lớn PP1 và PP2A. Consequently, the electrophilic residue of the Mdhb moiety remains capable of interactions with various cellular reaction partners (e.g., DNA, proteins), therefore, explaining the carcinogenic nature of NOD.

Previous analyses of field samples containing MC-RR variants via PPIA and other detection methods provided evidence that the protein-phosphatase-inhibiting (PPI) capađô thị of MC-RR variants was not of the expected potency (19-20). To clarify which of the MC-RR variants are responsible for this lowered PPI capađô thị, two commonly produced demethylated MCs (MC-RR & MC-RR) isolated from P. rubescens were characterized using HPLC-DAD, LC/MS, ELISA, PPIA, andimmunoblotting techniques.

Materials and Methods

Sampling và Cultivation of Cyanobacteria. Two unialgal Lake Ammersee P.. rubescens cultures were isolated from vertical seston samples (DS-129 & DS-29V, see the Sup-

porting Information). DS-129, DS-29V, & axenic strain A7 (Lake Zurich) were grown as batch cultures in 50 mL conical flasks at 21 °C và a 16:8h light:dark cycle with ca. 40pE/m2s from fluorescent tube lighting. Cells were harvested after 6 weeks of growth and stored at -trăng tròn °C. DS-29V, known khổng lồ exclusively produce MC-RR, was used khổng lồ purify -MC-RR. A7 was used lớn purify MC-RR.

Extraction and Purification. The extraction of MCs from strain A7 và DS-29V was carried out in triplicates with 70% MeOH with 15 min sonication (see the Supporting Information). After centrifugation (15 min, 3000g), supernatents were pooled, diluted to lớn less than 10% MeOH, and purified by Ci8 solid-phase extraction. C18 SPE cartridges (International Sorbent Technology, Germany) were conditioned with 100% MeOH (15 mL) followed by an equilibration step with deionized water (15 mL). Following application of the cyanobacterial extracts, the cartridge was washed with deionized water (15 mL) & 10% MeOH (15 mL), and the toxin was subsequently eluted with 50 mL 100% MeOH. This methanolic fraction was dried under a gentle stream of nitroren, resuspended in 100% MeOH, and finally diluted khổng lồ 20% MeOH with purified water. Separation và purification of the MCs was performed on a Cw Grom-Sil 1trăng tròn ODS-4, HE reversed phase column (11 pm particle kích thước, 250 x 10 milimet, Grom, Germany) using an isocratic 27% acetonitrile & 0.0135M ammonium acetate buffer (3 mL/min) as sản phẩm điện thoại phase (v/v). The HPLC system consisted of a pump solvent module Model 125 & an autosampler model 507e (Beck-man, Germany) coupled with a photodiode-array detector SPD-M10A (Shimadzu, Germany).

The methanolic (70%) extraction of MCs from strain DS-129 (5 mg DS-129 dw in 5 mL MeOH) was carried out in triplicates including sonication (15 min). The three super-natants were pooled after centrifugation (15 min, 3000g), diluted to lớn less than 10% MeOH and purified by C18 solidphase extraction as described above sầu (Scheme 1).

Analysis. RP-HPLC UV-DAD. AC reversed-phase column (Grom-Sil 1trăng tròn ODS-4 HE, 4.6 x 250 milimet, 5 pm particle kích cỡ, Grom, Germany) was used for MC chromatography. A solvent gradient with A (water) and B (acetonitrile), both containing 0.05% trifluoroacetic acid (TFA) (21), was established with a flow rate of 1 mL/min. MC-RR & the purified MC-RR variants were diluted in 20% MeOH for chromatographic analysis (determination of purity and quantification), and identified by their respective sầu retention times và characteristic UV absorption spectra.

For quantification of MCs, MC-LR was dissolved in 100% MeOH, diluted in 100% MeOH to lớn a concentration of 10 pM, followed by photometric concentration determination. Each sample was analyzed in triplicates, allowing calculation of the mean absorption. Haradaet al. (22) reported anidentical molar absorption coefficient (e) for MC-LR và MC-RR (e = 39 800 L mol-1cm-1). Lambert—Beer's law allowed calculation of the molar concentration of MC-LR and MC-RR. The molar concentration served lớn establish a MC-LR standard curve sầu in the RP-HPLC UV-DAD system. Concentrations of -MC-RR were calculated using standard curve sầu linear regression as no differences in e of MC-LR or MC-RR lớn MC-RR are expected based on structure comparison (22). For MC-RR an e= 50 400 L mol-1cm-1, established by Blom et al. (2), was used. To discriminate between MC-RR và MC-RR synthesized by DS-129, DS-129 extract was spiked with purified MC-RR from strain A7 (2).

HPLC-ESI-MS/LC/MS. Identification of MC-RR and MC-RR synthesized by DS-129 was achieved by recording mass spectra of the semi-purified MC-RR variants on a combined LC/MS (LCQ Duo mass spectrometer, Finnigan Thermoquest, U.S.) with an electrospray source (ESI-MS). The analysis was performed on a Shimadzu 10AVP

system with a photodiode-array detector, a C18 reversed-phase column (Grom-Sil 1đôi mươi ODS-4 HE, 4.6 x 250 milimet, 5pm particle form size, Stagroma, Germany), & a flow rate of 1 mL/ min. Solvent A (UV-treated deionized water) và solvent B (acetonitrile) were both acidified with 0.05% TFA (Fluka, Switzerland). A linear increase in three steps was applied (solvent B from 30 lớn 35% in 10 min, 35 lớn 70% in 30 min, 70-100% in 2 min, isocratic 8 min, (2).

Anti-Adda-ELISA. MC standards were analyzed with an anti-Adda-ELISA kit (23) in accordance with the manufacturer's instructions (Abraxis, U.S.). The assay was carried out three times in duplicate with MC-RR, MC-RR, & MC-RR. MC-LR was used as the internal standard.

cPPIA. The colorimetric protein-phosphatase-inhibition assay (cPPIA) determines the in vitro inhibitory capacity of MCs and NOD on protein-phosphatase activity (PP1 & PP2A). Tests were carried out with PP1 (rabbit skeletal muscle, recombinant (E. coli), New England Biolabs, U.S.) and PP2A (isolated from human red blood cells, V6311, Promega, U.S.) as previously described (24). p-Nitrophenylphosphate (pNPP., Acros, U.S.) was used as phosphatase substrate. The assay was carried out three times in duplicate with MCs và NOD (see the Supporting Information).

SDS-PAGE, Western Blotting (Anti-PP1 and Anti-Adda). To examine potential covalent binding of MC-LR, MC-RR, MC-RR, MC-RR, và NOD khổng lồ the catalytic subunit of PP1, MCs and NOD were incubated with PP1 (1 IU per 8 pL enzyme buffer) in enzyme buffer (50 mM Tris Base, 4 mM EDTA, pH 7.0, 1 mM phenylmethylsulpho-nylfluoride (PMSF), 1 mM benzamidine, 0.2% (v/v) ^-mer-captoethanol, 2% (v/v) Triton X-100) at 30 °C for 1.5 h. MCs and NOD (0.5 nmol per 8 pL enzyme buffer) were added in concentrations in excess to PP1 concentration present in the total reaction volume of 30 pL. Three independent replicate experiments were carried out.

To determine the MC-RR-PP1 covalent-binding reaction profile, MC-RR was co-incubated with the catalytic subunit of PP1, using the incubation protocol as already described above. This experiment was carried out in duplicate with seven different incubation times, ranging from 0 min khổng lồ 16h.

MC-PP1 Protein Complex Sample Preparation. At termination of incubation, MC-PP1 protein complexes were diluted 1:4 with SDS reducing sample buffer (1.5 M Tris Base; pH 8.8, 12.5% (v/v); glycerine, 10% (v/v); 10% SDS solution (w/v), 20% (v/v); ^-mercaptoethanol, 5% (v/v), bromphenolxanh, 1% (v/v)) và incubated at 95 °Cfor5min. Total protein concentrations were 0.17 units và 0.3 units protein-phosphatase employed for immunostaining with anti-PP1 and anti-Addomain authority antibody toàn thân (see below). To determine the molecular weight of the detected proteins, a full range recombinant protein molecular weight marker (10, 250 kD) was used (RPN 800, Amersham mê Pharmacia Biotech, Germany).

SDS-PAGE- Westernblotting(PP1 andanti-Adda). Proteins were separated using 10% SDS-Gel electrophoresis (25), at room temperature, 200 V và 35 min (Mini-PROTEAN II, Biorad, Germany). Polyacrylamide gels were equilibrated for 30 min in blotting buffer. Subsequently, proteins were blotted semi-dry onto nitrocelluthất bại membranes (PROTRAN, 0.2pm pore kích cỡ, Schleicher & Schuell, Germany). Westernblotting was carried out in a Mini-PROTEAN II for 90 min at 300 mA and 4 °C. Membranes were blocked in TTBS (Tween Tris buffered saline: 100 mM Tris-HCl, 0.9% NaCl (w/v), 0.1% Tween 20 (v/v)) containing 1% (w/v) bovine serum albumin for 30 min at room temperature. Primary antibodies, rabbit anti-PP1 (IgG fraction of antiserum, P7979, Sigma, Germany) và polyclonal sheep anti-Addomain authority no. 824 (23), were both diluted 1:1000 in TTBS. Equilibration was carried out for 60 min. After washing three times (15 min each), membranes were incubated with an alkaline phosphatase-conjugated

FIGURE 1. Analytical determinations of MC-RR variants in DS-129 extract. (a) HPLC chromatogram: arrows depict MC-RR and MC-RR (inserts show UV-absorption spectra of the two MC-RR variants); (b) LC/MS chromatogram & MS/MS fragmentation spectra of MC-RR (A) & MC-RR (B). Both MCs show fragment ions of m/z = 714 representing 1 ; (c) HPLC chromatograms (A = 239 nm) of MC-RR variants pre- & post-spiking with MC-RR.

horse anti-rabbit antibody toàn thân (Robịt Biochemicals, Germany) or rabbit anti-sheep antibody toàn thân (Sigma-Aldrich, Germany) as secondary antitoàn thân for 60 min. Membranes were washed three times for 15 min with TTBS & 15 min with TBS (100 mM Tris-HCl, 0.9% NaCl (w/v)). Alkaline phosphatase specific reactions were carried out using FastRed (F-4648, Sigma, Germany) as a substrate, và staining was stopped with purified water.

Results

HPLC-analysis of extracts of the Phường. rubescens culture originally isolated from Lake Ammersee (DS-129) provided for two main MC peaks (Figure 1a). These were identified both by their characteristic UV-spectra (Figure 1a, inserts) and their retention times in comparison lớn the MC-RR standard. The retention times of these two MCs differed only

marginally. Due to insufficient peak separation, the quantification of individual peaks và thus specific variants was imprecise. To confirm the identity of the detected MCs, samples were analyzed byLC/MS. For both MC peaks, double charged ions with the mass of m/z 513.2 Dalton 2+ were observed (Figure 1b). These ions are characteristic for three demethylated MC-RR congeners with the mass of 1023 Dalton, MC-RR, MC-RR, & MC-RR. To further identify the isolated MCs LC-MS/MS runs were carried out. Both mass spectra showed a significant fragment ion with the mass of m/z 714. This fragment ion may contain +. Thus, it can be assumed that the MCs of interest are MC-RR và MC-RR, as the fragmentation of MC-RR would result in a fragment with the mass of m/z 728 +. To discriminate between the two MC-RR variants, the extract of the Phường. rubescens strain DS-129 from Lake Ammersee was spiked with purified MC-RR from strain A7 (2), which resulted in a distinct increase of the second peak (Figure 1c), therefore confirming the presence of MC-RR in the second peak.

Quantification of MC-RR, from strain DS29V, & MC-RR, from strain A7, was achieved via analytical HPLC using published MC congener specific absorption coefficients (e,Table 1a). The loss of the methyl group at position 3 in MC-RR was not considered to lớn have any influence on e. Therefore, e = 39 800 Lmol-1cm-1 for MC-LR và MC-RR in MeOH (22) was used for concentration determinations of MC-RR. As MC-RR has a distinctly different e(50 400 L mol-1cm-1, (2), the following equation was used for the concentration determination of MC-RR via HPLC & MC-LR as standard:

c(Asp3,Dhb7>MC-RR) =

c(MC-LR) x e(MC-LR)/e (Asp3,Dhb7>MC-RR)

= c(MC-LR) x 39 800/50 400

= c(MC-LR) x С.79

Concentration determination for MC-RR; c = concentration; e = molar absorption coefficient. To support the above quantification results, MC-RR, MC-RR, & MC-RR were also analyzed by anti-Adda-ELISA. The antibody toàn thân used in this ELISA recognizes the Adda-group of all MC & NOD congeners that contain the Adda side-chain (23). Application of congener specific e (2, 22) for concentration determinations resulted in comparable MC-RR variant concentrations (Table 1a) when determined via HPLC-DAD or anti-Adda-ELISA (differences 80, ref 33), the broad spectrum of their chemical properties and the lack of analytical standards, accurate quantification

of single MCs is problematic. One of the currently acceptable methods is the photometric determination using HPLC-DAD. However, photometric determination depends on the accuracy và availability of the respective sầu absorption coefficients (e). Indeed, the eof MC-RR (2) và MC-LR/MC-RR (22) differ substantially, which potentially leads khổng lồ an overestimation of the concentration of MC-RR if the concentration of MC-RR is calculated using MC-LR or MC-RR as standards (Table 1a). Using MC-LR or MC-RR standard curves for quantification is, however, possible if a correction factor is introduced, as demonstrated in eq 1. Indeed, the comparison of quantification of MC-RR via HPLC-DAD (including correction factor) & via anti-Adda-ELISA demonstrated high determination consistency.

The MC-LR and MC-RR IC50 values determined with the cPPIA (Table 1b) areinagreementwithpreviouslypublished values achieved with comparable assays (34, 35). The distinctly higher IC50 values for MC-RR corroborate earlier reports by Blom et al. (36) using a fluorescent PPIA substrate with PP1 & PP2A.

In a study by Rapala et al. (20), the concentration of -MC-RR (isolated from strains from Finnish lakes) measured with the PPIA (using PP1) were only 5% of that determined by ELISA và HPLC. As demonstrated here, it is very difficult khổng lồ distinguish between MC-RR và MC-RR based on analyses using retention time, UV-spectra, & low-resolution mass spectra. Therefore, it is possible that MC-RR was one or even the predominant MC congener present in the sample analyzed by Rapala et al. (20), which would explain the discrepancy between the ELISA, HPLC, & the PPIA data reported. However, as NMR-studies and/or standards are essential khổng lồ indiscriminately identify MC-RR, which were not available lớn Rapala et al. (20), the latter interpretation cannot be corroborated. Thus the observation that quantification of MCs by HPLC-DAD & ELISA differ distinctly from quantification results obtained via PPIAs may be explained in part by MC congener inherent differences in PP.. inhibitory capathành phố. However, as not all PPIAs reported employed the same PPhường. source (native sầu tissue PPhường preparations of different tissue or animal origin or recombinant PPs), some of the differences observed ahy vọng the PP inhibitory capacity of MC congeners may also be due khổng lồ methodical variations and artifacts (Table 1b). The latter observations highlight the problems associated with the routine use of the PPIA lớn determine MC và NOD concentration measurements in water and cyanobacterial extract samples.

Moreover, it must be taken into tài khoản that the PPIA determines only the PP.. inhibition potential of a given sample and, therefore, only the sum of all PPhường inhibiting MCs in this sample. As it cannot a priori be assumed that the more than 80 different MC congeners inhibit PPs equally, as exemplified by the comparison of the PP1 & 2A inhibiting capathành phố of MC-RR & MC-RR in the study here, the results obtained with PPIAs may severely underestimate the actual MC concentrations present. However, not all Dhb7-contain-ing MC congeners demonstrate weaker PP.. inhibition capacity. Indeed, neither MC-HilR (37) nor MC-HtyR (10) were observed to have lower PP inhibitive sầu activity than corresponding concentrations of MC-LR (Table 1b). Conversely, other non-MC or -NOD PPhường. inhibitors present in water or bloom samples analyzed via PPIA may wrongly suggest the presence of PP inhibiting MCs or NOD. Thus the results of the cPPIAprovide amere estimate of PP-inhibiting potential but no proof of MC or NOD presence nor any information on the MC or NOD congeners present.

The results presented here in conjunction with evidence published earlier (10) suggest that MC congeners lacking a MDha on position 7 vị not covalently bind lớn PPs. This pertains not only lớn Dhb7 MCs, but also khổng lồ MCs with an alanine or serine at position 7 (summarized in Kaya et al. (38)). Despite that this study demonstrated that -MC-RR does not covalently bind lớn the catalytic subunit of PP1 in vitro (Figure 2c), và a similar observation was reportedfor MC-HtyRbyHastieetal. (10), the Dhb at position 7 và, therefore the lachồng of covalent binding capability to lớn PPs, does not a priori explain the weaker inhibition of the PPs, especially as no weaker PP inhibition was reportedfor Dhb7-containingMC-HilR (37) và MC-HtyR (10). From the currently available data it is thus hypothesized that the combination of the two arginines at positions 2 and 4 with Dhb at position 7 may be sterically hindering and thus restrict binding of MC-RR khổng lồ the catalytic subunit of the PP.

The numerous variations in amino acid composition and thus high number of MC congeners is of great concern, especially when in vivo adễ thương toxithành phố data are the sole information available and chronic data are scarce or absent. The consequence is that for better understanding of potential

risk acute in vivo data is compared lớn mechanistic in vitro data. Indeed, no difference in the LD50 values (mice, i.p.) can be observedfor MC-RR (27) and MC-RR (32). Furthermore, MC-RR was shown to be more toxic to lớn invertebrates (on the basis of LC50 values) than MC-LR or NOD (2). These observations suggest that the PP inhibiting capađô thị & the ability khổng lồ size covalent bonds lớn enzymes of the PPhường family bởi not consistently explain the observed in vivo toxithành phố và thus are a poor measure of potential risk. Furthermore, although the LD50 values of MC-LR & MC-LR differed only marginally (Table 1b), mice were treated once per week (i.p.) for 14 months with MC-LR presented with a distinctly reduced liver tumor incidence than mice treated with MC-LR (39,40). Whether a lower PPhường inhibiting capađô thị (toxicodynamics) or reduced biological availability (toxico-kinetics) of MC-LR is decisive for liver tumor promotion is yet to lớn be determined. Consequently, the presence of a Dhb group in a given MC congener, và therefore the laông chồng of covalent binding khổng lồ PPs, may be indicative for the tumor-promoting capađô thị of individual MCs, but not for its axinh đẹp toxithành phố.

The current risk assessment model for MC exposure is based entirely on toxicokinetic & -dynamic properties of MCs & especially on the subchronic toxiđô thị of MC-LR (8, 41). Drinking water/recreational water guideline values are then calculated based on MC-LR equivalents, primarily calculated based on the PPhường. inhibiting potential of the different congeners. Although PPs are assumed khổng lồ be the primary target for MCs và NODs, the caveats of determining PP. inhibitory capathành phố as a measure of potential in vivo toxiđô thị must be kept in mind. Indeed, it is becoming more apparent that the PP inhibiting capathành phố of most MCs is not directly correlated with their respective in vivo toxiđô thị (based on LD50 values).

Therefore, the PP.. inhibiting potencies presented here when compared to lớn the in vivo data available, strongly suggest that MC congeners can differ profoundly in their toxicody-namic and -kinetic properties and, therefore, emphasize, as suggested by Dietrich and Hoeger (8) earlier, that the risk assessment schemes based on MC-LR equivalents are insufficient.

Acknowledgments

Parts of this project were funded by the EU (QLRT-2001-02634, D.R.D. & S.H.) and the Arthur-and-Aenne-Feindt Foundation (D.R.D., B.E., & D.S.). Thanks khổng lồ Dr. Evelyn O'Brien & khổng lồ Prof. Dr. F. Juttner for helpful comments and for valuable discussions. S.J.H. and D.S. contributed equally khổng lồ this work.

Supporting Information Available

Further details about the chemicals and strains used in the experiment, a schematic ofthe experiment, và information about the sampling và cultivation of cyanobacteria. This material is available không lấy phí of charge via the Internet at http:// pubs.acs.org.

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Xem thêm: Trọng Tâm Của Tứ Diện Trong Không Gian, Độ Trọng Tâm G Của Tứ Diện Abcd A(D

Received for nhận xét November 8, 2006. Revised manuscript received January 25, 2007. Accepted January 26, 2007.


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