Environ. Sci. Technol. 2000, 34, 3372-3378
Hepatotoxin Production Kinetics of the Cyanobacterium Microcystis aeruginosa PCC 7820, as Determined by HPLC-Mass Spectrometry and Protein Phosphatase Bioassay C EÄ D R I C R O B I L L O T , † J O E¨ L L E V I N H , ‡ SIMONE PUISEUX-DAO,§ AND M A R I E - C L A I R E H E N N I O N * ,† Laboratoire Environnement et Chimie Analytique CNRS 657 and Laboratoire de Neurobiologie et Diversite´ Cellulaire CNRS UMR 7637, Ecole Supe´rieure de Physique et de Chimie Industrielles de la Ville de Paris, 10 rue Vauquelin, 75231 Paris Cedex 05, France, and Laboratoire de Cryptogamie, Muse´um National d’Histoire Naturelle, 12 rue Buffon, 75231 Paris Cedex 05, France
Freshwater cyanobacteria (blue-green algae) can produce numerous potent toxins and represent an increasing environmental hazard. The microcystin content (cyclic heptapeptidic toxins) of the hepatotoxic cyanobacterium Microcystis aeruginosa PCC 7820 was investigated. Ten microcystins were identified using high performance liquid microchromatography (micro HPLC) coupled to either a ultraviolet (UV) diode-array detector (DAD) or an electrospray ionization (ESI) mass spectrometer. Three new variants were identified: desmethylated microcystin LW (mcyst-dMeLW), desmethylated microcystin LF (mcystdMeLF), and microcystin LL (mcyst-LL) by CollisionInduced Dissociation/Post-Source Decay Matrix-assisted Laser Desorption/Ionization-Time-of-Flight mass spectrometry (CID/PSD MALDI-TOF MS). The concentration of intracellular microcystins reached 2-8 mg/g of dried cells, with a microcystin LR (mcyst-LR) equivalent of 1-5 mg/g as estimated by protein phosphatase 2A (PP2A) inhibition assay. Toxin production can be correlated to biomass increase up to the middle of the exponential phase of growth and ceases thereafter. Toxin release occurred during the stationary phase, and extracellular microcystin concentration reached 0.25 mg/L. Intracellular microcystin pool composition (MPC) was constant with 51 ( 2% mcystLR, whereas this toxin stood for only 29 ( 3% of extracellular MPC. Mcyst-LR, the less hydrophobic microcystin, diffuses less easily across membranes. Hydrophobicity might play a key role in microcystin release process.
Introduction Water blooms of toxic Cyanobacteria (blue-green algae) are commonly found in eutrophic freshwater and represent an increasing environmental hazard because of the potent toxins they can produce (1, 2). The most frequently reported toxins are hepatotoxins, the largest group being heptapeptides, known as microcystins (mcyst). More than 60 mcyst have already been isolated from cyanobacterial species (3). They 3372
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are presumed to be synthesized nonribosomally by peptide synthetases (4, 5). Their common cyclic structure is denoted by cyclo(-D-Ala-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where MeAsp stands for erythro-β-methylaspartic acid, Mdha for N-methyldehydroalanine, Adda for 3-amino-9-methoxy2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, and X and Z for two variable amino acids. These two residues are used to name the molecule (Figure 1); for example, microcystin LR (mcyst-LR) contains Leucine (X ) L) and Arginine (Z ) R). Minor modifications can occur on the other amino acids, such as demethylation on MeAsp or Mdha, or esterification of glutamic acid. These compounds are responsible for the death of fish, birds, and wild and domestic animals all over the world (6, 7). Furthermore, adverse effects on human health have been recognized (8, 9). Mcyst are potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) from animals and higher plants (10, 11) and may consequently act as tumor promoters at the nanomole level (10-13). However, not all species synthesize toxins, and the potency of blooms can vary substantially according to sites, seasons, weeks, or even days. These variations could be due to changes in species composition, to the production of different toxins with varying toxicity for each clone, and to the influence of environmental factors. The genetic and biochemical regulation of mcyst production is not yet understood. Environmental factors such as light, temperature, pH, nutrients, and trace metals are known to affect in vitro toxin production and growth of toxic strains of M. aeruginosa (14-18). Here we present the study of the toxin production kinetics of M. aeruginosa PCC 7820 during growth. Biomass and toxin production were measured along with growth in controlled cultures. Microliquid chromatography (20) was used to separately analyze the production of intracellular and extracellular microcystins. The detection was performed both with a photodiode-array detector (DAD) and with a mass spectrometer coupled via an electrospray ionization interface for the identification of mcyst. MALDI-TOF mass spectrometry in the CID/PSD mode has been recently applied to identify a new cyanobacterial metabolite called anabaenopeptin (21). In our study, this technique allowed the complete identification of three microcystins detected for the first time in Microcystis aeruginosa PCC 7820 cultures: desmethylated microcystin LW (mcyst-dMeLW), desmethylated microcystin LF (mcyst-dMeLF), and microcystin LL (mcyst-LL). The two first are new microcystin variants. Intracellular mcyst concentrations measured by HPLC were compared with those obtained by PP2A inhibition assay. Finally the intracellular microcystins pool composition (MPC) and the extracellular MPC were investigated, and some physicochemical factors which could influence toxin release were determined.
Materials and Methods Cyanobacterium Culture. Cultures of Microcystis aeruginosa PCC 7820, supplied by Institut Pasteur (Paris, France), were maintained in sterilized 250 mL Erlenmeyer flasks containing 100 mL of BG110 medium (+2 mM NaNO3) at 25 °C under white light (1900 lux, LD 16-8 h). Sterile air was bubbled through the culture medium. Each measure of biomass and * Correspondingauthorphone: 33.1.40.79.46.51;fax: 33.1.40.79.47.76; e-mail:
[email protected]. † Laboratoire Environnement et Chimie Analytique, Ecole Supe´rieure de Physique et de Chimie Industrielles de la Ville de Paris. ‡Laboratoire de Neurobiologie et Diversite ´ Cellulaire, Ecole Supe´rieure de Physique et de Chimie Industrielles de la Ville de Paris. § Muse ´ um National d’Histoire Naturelle. 10.1021/es991294v CCC: $19.00
2000 American Chemical Society Published on Web 07/06/2000
FIGURE 1. Structure of microcystins. toxin production was associated with one flask. Cells and culture media were separated by filtration through 47 mm GF/C disks (Whatman, Maidstone, Kent, U.K.). Samples Preparation. The disks, with attached cyanobacterial cells, were dried at 50 °C for 12 h, and the dried cellular mass was measured. Cells were then sonicated for 5 min and extracted in methanol for 15 min. The extraction procedure was repeated three times. After filtration, the sample was evaporated to dryness at 45 °C under reduced pressure. The residue, called intracellular extract, was then dissolved in 800 µL methanol and stored at -20 °C prior to analysis. Aqueous filtrates were preconcentrated on a Bakerbond SPE cartridge (Baker, Deventer, Netherlands) packed with 500 mg of octadecylsilica. The solid-phase extraction procedure (22) involved (i) activation of the cartridge with 5 mL of methanol, (ii) conditioning with 10 mL of Milli-Q water, (iii) percolation of the sample at a flow-rate of 10 mL/min, and (iv) cleanup with 5 mL of 20% aqueous methanol. Air was then sucked up through the cartridge for 2 min. Analytes were subsequently desorbed with 3 mL of methanol. The residue, called extracellular extract, was evaporated to dryness at 45 °C under reduced pressure, dissolved in 200 µL of methanol, and stored at -20 °C prior to analysis. Microliquid Chromatography. The instrumentation for micro HPLC consisted of two LC10AS pumps (Shimadzu, Kyoto, Japan) connected to a homemade 1/16 flow splitter. The outlet was linked to a Kontron automatic sampler sixport valve with external sample loop of 2 µL. UV detection was performed at 238 nm with a SPD-10A detector (Shimadzu) equipped with a 160 nL U-shaped microcell (LC Packings, Amsterdam, Netherlands) and also with a SPDM10A photodiode-array detector (Shimadzu) equipped with a 160 nL U-shaped microcell (prototype LC Packings). Separation of cyanobacterial samples was performed on a Hypersil BDS C18 column (250 × 1 mm i.d., 5 µm) at a flowrate of 50 µL/min with a mixture of Milli-Q water acidified to pH 2.5 with trifluoroacetic acid (solvent A) and pure acetonitrile (solvent B), using the following linear gradient: 30% of B at 0 min, 50% of B at 40 min, 80% of B at 60 min, and 100% of B at 65 min. Mass Spectrometry. For HPLC-ESI-MS experiments, the outlet of the microcolumn was fitted to the stainless steel needle of a ESI source mass spectrometer Platform (VG Biotech, Fisons Instruments, Altrincham, U.K.). The capillary voltage was adjusted to 3.5 kV. To obtain a partial fragmentation of mcyst, the cone voltage was adjusted to 39 V. The analyzer was calibrated from m/z 600 to 1200 in positive-ion mode with poly(ethylene glycol). MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time Of Flight) mass spectra were recorded in the CID/ PSD mode with a single stage reflectron MALDI-TOF mass spectrometer (Voyager STR, PerSeptive Biosystems, Inc., Framingham, MA) equipped with a delayed extraction device (23). A collision cell was installed just behind the ion source,
for the CID/PSD mode. The precursor ions were selected with a Time Ion Selector with a mass window of approximately 30 m/z units to maximize the transmission rate. A nitrogen laser beam (λ ) 337 nm, 3 ns-wide pulse at 20 Hz, laser power set just above the desorption threshold) was focused for desorption on the stainless steel target, and the ions were detected by the dual channel plate reflector detector after a total flight length of 3 m. Delayed extraction time was set at 350 ns. The accelerating voltage was set at 25 kV with an intermediate grid voltage of 18 kV and a guide wire voltage of 25 V for the precursor. At least 100 shots were averaged for each mass range acquired before generating the PSD spectrum under the control of the Voyager and Grams software. In the CID/PSD mode argon was chosen as the collision gas with a pressure of about 10-6 mbar. Samples were prepared with the dried droplet method by mixing 0.5 µL of sample and 0.5 µL of a saturated solution of 2,6dihydroxybenzoic acid in 1:9 (v/v) ethanol/aqueous ascorbic acid (50 mg/µL) directly on the sample plate. External calibration was performed with a mixture of des-Arg1Bradykinin, neurotensin, and ACTH (18-39 clip), with monoisotopic m/z masses for [M + H]+ of 904.47, 1672.92, and 2465.20 Da, respectively. Protein Phosphatase 2A Inhibition Assay. PP2A inhibition assay for okadaic acid (24) has been adapted to microcystins by C. Rivasseau et al. (25) and N. Bouaı¨cha. The assay is based on the degradation of p-nitrophenyl phosphate (uncolored) into p-nitrophenol (yellow). Inhibition rate is directly related to the absorbance at 405 nm measured by a plate-reader BIO-TEK (Winooski, VT). Mcyst concentration was obtained by reporting the inhibition rate on a standard mcyst-LR calibration curve. Reagents and Standards. All reagents were of HPLC grade or analytical grade. Methanol was purchased from Carlo Erba (Milan, Italy), acetonitrile from Baker (Phillipsburg, NJ). LCquality water was obtained by purifying demineralized water in a Milli-Q filtration system (Millipore, Bedford, MA). p-Nitrophenyl phosphate and also mcyst-LR were purchased from Sigma (Saint Louis, MO). PP2A extracted from human hepatocytes was obtained from Euromedex (Souffelweyersheim, France).
Results and Discussion Identification and Quantitation of Microcystins Produced by Microcystis aeruginosa PCC 7820 in Cultures. Cellular extracts and growth medium were analyzed by HPLC-DAD. Microcystins all contain the Adda residue which includes two conjugated π-bonds responsible for a ultraviolet (UV) absorbance maximum at 238 nm so that their UV spectra are very characteristic. The UV spectra of tryptophan and tyrosine containing mcyst can present an additional absorbance peak at about 223 and 232 nm, respectively. HPLC-DAD analyses revealed the presence of five major peaks that can be attributed to microcystins. Two of them contain a tryptophan group as demonstrated by an additional absorbance peak at 223 nm (Figure 2). This method allows a rapid detection of these compounds but is not informative enough to identify each microcystin. A LC-ESI-MS coupling was set up to achieve the identification. The electrospray interface is very suitable for microcystin ionization. Their masses range from 900 to 1100 Da. These compounds can easily lose a characteristic group [PhCH2CH(OMe)] (inducing a loss of 134 Da), resulting from the alpha-cleavage of the methoxyl group of the Adda residue. This allows the identification of microcystins with certainty using the HPLC-ESI-MS method, as shown in Figure 2. The spectra present the signals associated with the pseudo molecular ion [M + H]+ and with the remainder of the peptide after cleavage [M + H - 134]+. This analysis confirmed the presence of mcyst-LR, methylated [D-Glu(OCH3)]mcyst-LR, VOL. 34, NO. 16, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. HPLC chromatogram of Microcystis aeruginosa PCC 7820 cellular extracts monitored at 238 nm; ultraviolet spectra and electrospray mass spectra associated to (A) mcyst-LR and mcyst-dMeLR, (B) mcyst-dMeLW, and (C) mcyst-LW and mcyst-dMeLF. mcyst-LY, mcyst-LM, mcyst-LW, and mcyst-LF described by Lawton et al. (26, 27). One minor compound was identified as desmethylated mcyst-LR (mcyst-dMeLR); it coeluated with mcyst-LR as already described by Bateman et al. (28). Furthermore, a new tryptophan-containing microcystin at 1011 Da was identified as desmethylated mcyst-LW (mcystdMeLW). Two other compounds were detected at 972 and 953 Da, respectively. The compound at 972 Da coeluated with mcyst-LW. This molecular mass could either correspond to mcyst-VF as already described (28) or to desmethylated mcyst-LF (mcyst-dMeLF). The peak containing both this microcystin and mcyst-LW was collected. The other compound at 953 Da coeluated with mcyst-LF. This molecular mass could either correspond to mcyst-AR as already described (28) or to mcyst-LL that is more consistent with the hydrophobicity of this type of molecule. As a matter of fact arginine-containing microcystins are much more polar than mcyst-LF. The peak containing both this microcystin and mcyst-LF was collected. We used CID/PSD MALDI TOF mass spectrometry to further characterize the two unknown microcystins. MALDI TOF mass spectrometry analysis gave two monoisotopic molecular masses at 972.5 and 952.5 Da, respectively. In the low mass range CID/PSD MALDI TOF mass spectrometry produces immonium ions that are amino acid specific (29). For the microcystin at 972.5 Da, immonium ions associated to Leucine at 86 Da and Phenylalanine at 120 Da were observed, but no significant signal could be obtained at 72 Da for Valine. In analogy with previous tandem mass spectrometry studies (27), a characteristic fragment identified as [Leu-Ala-Mdha-Glu-C11H14O + H]+ was detected at 559.4 Da (see Figure 3A). No signal for [Val-Ala-Mdha-Glu-C11H14O + H]+ at 545.3 Da was observed. Table 1 summarizes the other fragments. The whole set of data confirmed the presence of desmethylated mcyst-LF rather than mcyst-VF as suggested in a previous study (28). For the microcystin at 952.5 Da, immonium ion associated to Leucine at 86 Da was observed, but no significant signal could be obtained at 129 Da for Arginine immonium ion and all related ions. Moreover the characteristic fragment identified as [Leu-Ala-Mdha-Glu3374
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C11H14O + H]+ was detected at 559.1 Da (see Figure 3B). No signal for [Ala-Ala-Mdha-Glu-C11H14O + H]+ at 517.3 Da was observed. Table 1 summarizes the other fragments. The whole set of data confirmed the presence of mcyst-LL rather than mcyst-AR as suggested in a previous study (28). The identification of mcyst-dMeLW was confirmed with the same strategy (CID/PSD spectrum not shown). The analytical tools set up in this study allowed the complete identification of 10 different microcystins in Microcystis aeruginosa PCC 7820 cultures, including three new variants: mcyst-LL, mcyst-dMeLW, and mcyst-dMeLF. Mcyst-dMeLW and mcyst-dMeLF have never been identified in any cyanobacterial culture. Moreover, a desmethylated variant at low concentration was observed for the three major microcystins mcyst-LR, mcyst-LW and mcyst-LF; are these variants byproducts of usual mcyst or not? Our results suggest that desmethylated variants might be derived from usual mcyst, undergoing an intracellular modification. These variants were detected in both intracellular and extracellular extracts, ruling out the hypothesis that desmethylated mcyst might originate from a degradation process in the culture medium. However, the possibility of a distinct path of synthesis cannot be excluded. During a toxic bloom in a french pond, Planktothrix agardhii cells were harvested and two major intracellular compounds were identified as mcystdMeRR and mcyst-dMeLR, whereas mcyst-RR and mcystLR could not be detected. The cyanobacterium Anabaena flos-aquae strain NRC 525-17 produces mcyst-dMeLR but no mcyst-LR (30). Further experiments have to be carried out to unterstand the origin of desmethylated variants. The total amount of mcyst was estimated by measuring the area of the four major UV absorption peaks at 238 nm, mainly corresponding to mcyst-LR, mcyst-LY, mcyst-LW, and mcyst-LF. But only three commercial standards (mcyst-LR, mcyst-YR, and mcyst-RR) were available implying that theoretically, only mcyst-LR could be quantified. However, the conjugated π-bonds on the Adda fragment, common to all Mcyst, are responsible for their UV absorption characteristics. So we approximated that UV absorbance coefficient at 238 nm is identical for all mcyst, as it has been verified
FIGURE 3. MALDI-TOF mass spectra recorded in CID/PSD mode of (A) mcyst-dMeLF and (B) mcyst-LL.
TABLE 1. Calculated Monoisotopic m/z Ratios of Specific Fragment Ions for Different Microcystins: Mcyst-dMeLF, Mcyst-VF, Mcyst-LL, and Mcyst-ARa m/z values
b
assignment
dMeLFb (X ) L, Y ) F)
VF (X ) V, Y ) F)
LLb (X ) L, Y ) L)
AR (X ) A, Y ) R)
[M + H]+ loss of PhCHdCH(OMe) PhCHdCH(OMe)+ [Ala-Mdha + H]+ [Mdha-Glu + H]+ [Mdha-Glu-C11H14O + H]+ [Y-MAsp-X-Ala-Mdha + H]+ [Y-MAsp-X-Ala-Mdha-Glu + H]+ [Y-MAsp-X-Ala-Mdha-Glu-C11H14O + H]+ [Y-Asp-X-Ala-Mdha + H]+ [Y-Asp-X-Ala-Mdha-Glu + H]+ [Y-Asp-X-Ala-Mdha-Glu-C11H14O + H]+ [X-Ala-Mdha-Glu-C11H14O + H]+ [X-Ala-Mdha-Glu + H]+
972.5 (972.5) 838.4 (838.3) 135.1 (135.2) 155.1 (154.9) 213.1 (213.3) 375.2 (375.1)
972.5 838.4 135.1 155.1 213.1 375.2 530.3 659.3 821.4
952.5 (952.5) 818.5 (818.3) 135.1 (134.9) 155.1 (155.0) 213.1 (213.0) 375.2 (375.0) 510.3 (510.1) 639.3 (639.2) 801.4 (801.5)
953.5 819.4 135.1 155.1 213.1 375.2 511.3 640.3 802.4
545.3 383.2
559.3 (559.1) 397.2 (397.0)
517.3 355.2
530.3 (530.1) 659.3 (659.2) 821.4 (821.5) 559.3 (559.4) 397.2 (397.7)
a Immonium ions of residues Ala, Val, Leu, Asp, Glu, Masp, Phe, and Arg have a m/z ratio of 44, 72, 86, 88, 102, 102, 120, and 129, respectively. Experimental m/z ratios of fragment ions for microcystins isolated from Microcystis aeruginosa PCC 7820 are indicated in brackets.
for the three available standards. Thus quantitations were made using the mcyst-LR calibration curve, though we had to admit a systematic error in the case of tryptophan and tyrosine containing mcyst, because of additional absorbance peaks, which probably modify their absorption coefficients at 238 nm. Intracellular mcyst concentration was studied by HPLC but also by the protein phosphatase 2A (PP2A) inhibition assay. In vivo, mcyst toxicity is based on the inhibition of serine-threonine phosphatases and the PP2A bioassay exploits this property. Though this does not take into account metabolisation processes, this enzymatic assay gives an estimation of the sample toxic potency whereas HPLC
supplies us with individual and total mcyst concentrations. Such a difference is crucial since mcyst present extremely different toxicities in vivo: the DL50 measured by intraperitoneal injection in mice of some mcyst produced by Microcystis aeruginosa are listed in Table 3. Therefore we expected relevant differences between the concentrations measured by HPLC and by the PP2A assay. The following ratio (called R) is an important parameter to consider since it is directly linked to the composition of the Mcyst pool.
R)
[equivalent mcyst - LR] by PP2A [total mcyst] by HPLC
Its value would theoretically be equal to 1 if the sample VOL. 34, NO. 16, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Microcystins Isolated from Microcystis aeruginosa PCC 7820 our study
mass (monoisotopic)
1 LR 2 (D-Asp3, Dha7, dMeAdda5)LR 3 Dglu(OCH3)LR 4 LY 5 LM 6 dMeLW 7 dMeLF 8 LW 9 LL 10 LF
994.5 980.5 1008.5 1001.5 969.5 1010.5 971.5 1024.5 952.5 951.5 985.5
Lawton et al. (26, 27) LR
Bateman et al. (28) LR (D-Asp3, Dha7, dMeAdda5)LR
Dglu(OCH3)LR LY LY LM
LW
VF LW AR
LF
LF
TABLE 3. DL50 of Some Microcystins Produced by Microcystis aeruginosa PCC 7820 microcystin
DL50 (µg/kg, i.p. mice)
ref
LR (D-Asp3)LR (Dha7)LR (dMeAdda5)LR LY AR
50 200-500 200-250 90-100 90 250
(31) (30) (31) (31) (32) (31)
contained only hepatotoxins that inhibit PP2A as much as mcyst-LR. Since mcyst-LR is the most toxic microcystin already tested in vivo, R is generally lower than 1. Our samples contain at least 10 microcystins, and several of them are less toxic in vivo than mcyst-LR (Table 3). As a result, in our case, R is expected to be inferior to 1. This parameter will be studied in detail in the following section about toxin production kinetics. Toxin Production Kinetics. Biomass was measured during Microcystis aeruginosa PCC 7820 growth. Triplicate measures were performed in the exponential phase and the beginning of the stationary phase. A two-phase growth without any latency period was observed (Figure 4a). Replication time was 2.6 ( 0.1 days, and the stationary phase was reached after 12 days, with a maximum cellular dry weight of approximately 60 mg (corresponding to 600 mg/L). We checked the high correlation between cellular density and dry weight and the good reproducibility of growth curves (data not shown). First, toxin production was measured by HPLC-UV, using the criteria defined above. Toxin amounts can be expressed as a function of time or biomass. The biomass is the best parameter signing for the state of the culture. Figure 4b shows the variations of total intracellular and extracellular mcyst amounts as a function of cellular dry weight. The extracellular mcyst amount increased at the beginning of the exponential phase and became stable at a dry weight of 20 mg. Then it increased strongly when the culture reached the stationary phase. This change can be explained by the release of toxins in the medium corresponding with the death and lysis of cells when they are out of nutrients. Intracellular mcyst amount increased linearly at the beginning of the growth, which means that cells produce mcyst in proportion with their biomass. From a dry weight of 30 mg, toxin production decreased and the Mcyst amount became stable, while an augmentation of the total cellular biomass was still observed for several days. This situation is well reflected in Figure 4c which shows a decrease of cell toxin content with time after day 7. This implies that in conditions where the nutrients become limiting, toxin production is drastically diminished and thus is not a priority for the cyanobacterium. Afterward, 3376
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FIGURE 4. Microcystis aeruginosa PCC 7820: (a) growth curve, (b) (9) extracellular and (0) intracellular total mcyst amount as a function of biomass, and (c) intracellular mcyst concentration as a function of time as measured by (9) HPLC and (0) PP2A assay. during the stationary phase, cells release their content into the medium leading to the decrease of intracellular mcyst amount. Regarding our results, toxin production does not seem to be a response to environmental stimuli, but, on the contrary, it probably forms part of Microcystis aeruginosa secondary metabolism. Intracellular mcyst concentration is an important parameter to consider when estimating the cell toxic potency. Its value as a function of time reaches a maximum at day 7, as shown in Figure 4c. These experiments demonstrate the key role of kinetics as far as toxin production is concerned. Total intracellular mcyst concentration varied from 2 to 8 mg/g depending on the day of analysis. Therefore different toxic species or strains can be compared in terms of toxin production only in association with their production kinetics. Intracellular mcyst production was also followed by the PP2A inhibition assay (Figure 4c). The variations are consistent with those observed by HPLC, with a maximum at day 7. However, the maximum concentration measured by the PP2A assay is lower than that measured by HPLC, with 5.1 ( 0.6 mg mcyst-LR equivalent/g from PP2A assay and 7.6 ( 1.0 mg/g from HPLC analysis. To understand these differences, a correlation diagram was established, displaying the mcyst concentration from PP2A assay as a function of the mcyst concentration from HPLC analysis (Figure 5). The diagram has a linear trend; the slope corresponds to the ratio R described previously. The calculated value is R ) 0.67 ( 0.03 and is lower than 1 as expected. Such a linear trend suggests that R is not time-dependent indicating that the mcyst pool might keep the same composition over the growth.
with 10 mL of a mother culture in stationary phase which contains a non-negligible amount of mcyst (about 2 µg). This source of mcyst has to be taken into account in our study. Therefore at day 0, the extracellular MPC was that of the mother culture in stationary phase, with a great proportion of mcyst-LR (>40%). During the exponential phase, we observe the sum of two contributions: that of the mother culture and that of the growing cells. Finally, during the stationary phase, cells release their whole content, and the extracellular MPC is similar to the intracellular MPC. FIGURE 5. Correlation between intracellular mcyst concentrations measured by HPLC and by the PP2A assay (r ) 0.74; n ) 14).
TABLE 4. Intracellular Mcyst Pool Composition (MPC) and Extracellular MPCnorm microcystin LR LY LW LF a
intracellular MPC (%) 51.0 8.6 21.0 19.4
Standard deviation (n ) 15).
(2.4)a (0.4) (0.4) (1.3) b
extracellular MPCnorm (%) 29 16 33 22
(3)b (4) (1) (3)
Standard deviation (n ) 3).
FIGURE 6. Extracellular mcyst-LR proportion as a function of time. HPLC analysis confirmed this hypothesis, showing that the intracellular mcyst pool composition (MPC) was very stable over time: the proportion of intracellular mcyst-LR varied between 50.1 and 51.5% during growth. Average values are listed in Table 4. This result implicates that when cells reduced toxin production around day 7, it was equally reduced for each microcystin. We can thus hypothesize that either all the microcystins have a common single production system or that they are produced by different systems with a common regulation pathway. Comparison of Extracellular and Intracellular Toxin Production. We studied intracellular mcyst pool composition (MPC) and extracellular MPC. The intracellular MPC has been analyzed previously but the evaluation of extracellular MPC is more complex. In fact, extracellular MPC was not constant, and its variation with time is shown in Figure 6. First it decreased and then increased with a pattern which can be related to the two growth phases: the exponential phase and the stationary phase. Indeed, during the exponential phase, cells produced intracellular mcyst, a small part of which being regularly released into the mediumsthe total intracellular mcyst amount could be more than 6-fold higher than the total extracellular mcyst amount. Then, during the stationary phase, lysis of many cells induced the release of their toxin content without any specificity, the total extracellular mcyst amount increased strongly, and the extracellular MPC drew near to the intracellular MPC. The detected decline during the first 7 days can be explained as follows: we have inoculated 100 mL of medium
To evaluate the contribution to extracellular MPC of growing cells only, we expressed two hypotheses: (i) mcyst degradation is negligible on the duration of the experiments, regarding the relatively soft conditions employed (pH 8.4, 25 °C) and the axenicity of the culture and (ii) the release of toxin in the extracellular medium is negligible during the first 3 days. So we normalized the measurement of each mcyst quantity by subtracting the corresponding approximative initial value. This eliminates the contribution of the mcyst introduced within the mother culture. This normalized extracellular MPC was called extracellular MPCnorm. Until the stationary phase, the extracellular MPCnorm corresponds to the actual toxin fraction released by the cells. The intracellular MPC and extracellular MPCnorm are reported in Table 4. McystLR was the major toxin (51 ( 2%) in cells, but its proportion was significantly lower in the culture medium (29 ( 3%). Mcyst-LR behaved differently from the three other main microcystins whose relative proportions stayed quite similar in intracellular and extracellular extracts. This suggests a difference in terms of membrane crossing in the case of mcyst-LR. The four main microcystins produced by the strain are mcyst-LR, mcyst-LY, mcyst-LW, and mcyst-LF. Mcyst-LR has one important particular characteristic: it is less hydrophobic than the three others with the lowest retention time in reversed phase HPLC, as confirmed by logP values of 2.16, 2.92, 3.46, and 3.56 for mcyst-LR, mcyst-LY, mcyst-LW, and mcyst-LF, respectively (33). The process of mcyst release in the aqueous medium is not well-known. Our results are in favor of a passive diffusion mechanism since the less hydrophobic molecule diffuses less easily across the membranes. The analytical tools set up in this study and more especially mass spectrometry techniques allowed the complete identification of 10 microcystins produced by Microcystis aeruginosa PCC 7820 including three new variants: mcyst-LL, mcyst-dMeLW, and mcyst-dMeLF. MALDI-TOF-MS in CID/ PSD mode is actually a promising technique for the structural analysis of this class of molecules. Micro-HPLC and PP2A inhibition assay gave consistent and complementary results for the estimation of strain toxinic content. The PP2A assay is performed as a screening technique followed by HPLC as a confirmation. This strategy appears to be very efficient for the environmental monitoring of mcyst. The extensive study of toxin production kinetics showed that mcyst present a common regulation pathway which is probably involved in the cyanobacterium secondary metabolism. The intracellular mcyst concentration was not constant. It implies that the comparison of toxin production between different species or strains should take into account their production kinetics. A massive release of toxins in the extracellular medium due to cell lysis has been observed. This phenomenom, associated to the relative stability of Mcyst in the aqueous medium, can induce water contamination in situ. Thus, regarding their strong tumor promoting activity, the monitoring of mcyst in freshwater ponds seems to be critical in environment and health management. VOL. 34, NO. 16, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Acknowledgments The authors would like to thank N. Tandeau de Marsac and R. Rippka from the Institut Pasteur for the gift of Microcystis aeruginosa PCC 7820, C. Rivasseau, J. P. Le Caer, and L. Via Ordorika for their friendly assistance, and finally G. Codd for helpful advice.
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Received for review November 17, 1999. Revised manuscript received May 12, 2000. Accepted May 15, 2000. ES991294V
