The Cyanobacterial Toxin Cylindrospermopsin Inhibits Pyrimidine Nucleotide Synthesis and Alters Cholesterol Distribution in Mice

M. Reisner*, S. Carmeli{dagger}, M. Werman{ddagger} and A. Sukenik§,1

* Department of Food Engineering and Biotechnology, Technion, Israel Institute of Technology, Haifa 32000 Israel; {dagger} School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel; {ddagger} Ministry of Health, District Health Office, Public Health Laboratory, P.O. Box 9526, Haifa, 35055, Israel; and § Israel Oceanographic and Limnological Research, Yigal Allon Kinneret Limnological Laboratory, P.O. Box 447, Migdal Gigdal 14950, Israel

Received April 29, 2004; accepted July 12, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hepatotoxin Cylindrospermopsin, a sulfated-guanidinium alkaloid with substituted dioxypyrimidine (uracil) moiety, was isolated from several cyanobacteria species. Our previous studies on the toxicity of cylindrospermopsin and its derivatives suggested that the uracil moiety is crucial for the toxicity and that such toxicity could partly stem from competitive binding of the toxin to a catalytic site(s) involved in the synthesis of pyrimidine nucleotides (i.e., uridine). In the present study we demonstrated that cylindrospermopsin inhibited in a noncompetitive manner the in vitro activity of uridine monophosphate (UMP) synthase complex (responsible for the conversion of orotic acid to UMP) in a cell free liver extract from mice, with an inhibition constant, KI, of 10 µM. Exposure of mice to cylindrospermopsin at subacute concentrations, via drinking water, only slightly affected the in vitro activity of UMP synthase. The typical metabolic disorder associated with the inhibition of UMP synthase activity, known as "orotic aciduria," was not observed under these conditions, but other anomalous metabolic responses related to cholesterol metabolism were developed.

Key Words: hepatotoxin; cyanobacteria; cylindrospermopsin; UMP synthase; cholesterol.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hepatotoxin cylindrospermopsin, a sulfated-guanidinium alkaloid with 5-susbtituted-2,4-dioxypyrimidine (uracil) moiety (Banker et al., 1997Go; Harada et al., 1994Go; Ohtani et al., 1992Go), was isolated from several cyanobacteria species, Cylindrospermopsis raciborskii (Woloszynska) (Ohtani et al., 1992Go), Umezakia natans (Harada et al., 1994Go), and Aphanizomenon ovalisporum (Forti) (Banker et al., 1997Go; Shaw et al., 1999Go). Blooms of cylindrospermopsin-producing cyanobacteria in lakes and fresh water reservoirs were reported in Australia (Ohtani et al., 1992Go; Shaw et al., 1999Go), Hungary (Vasas et al., 2002Go), Israel (Banker et al., 1997Go), Thailand (Li et al., 2001Go), Germany (Fastner et al., 2003Go), New Zealand (Stirling and Quilliam, 2001Go), and Japan (Harada et al., 1994Go). In few cases these blooms were associated with human morbidity and cattle mortality (Griffiths and Saker, 2003Go; Hawkins et al., 1985Go; Thomas et al., 1998Go); therefore they present a threat to water consumers worldwide.

Cylindrospermopsin was shown to be a potent inhibitor of protein synthesis. Terao et al. (1994)Go described the liver as the main target of this phycotoxin, with four consecutive phases of the pathological changes in the liver: protein synthesis inhibition, membrane proliferation, fat droplet accumulation, and cell death. Runnegar et al. (1994Go, 2002Go) found that cylindrospermopsin exposure led to a decrease in the content of reduced glutathione (GSH) in rat hepatocytes. The reduction in GSH induced by cylindrospermopsin was attributed to the inhibition of GSH synthesis rather than to increased consumption (Runnegar et al., 1995Go). Norris et al. (2002)Go suggested that activation of cylindrospermopsin by cytochrome P450 is of primary importance in the mechanism of action.

The nucleotide structure of cylindrospermopsin and the presence of potentially reactive guanidine and sulfate groups suggest that the toxin may exert its effect through an interaction with DNA or RNA. Covalent binding of cylindrospermopsin or its metabolites to DNA has been reported in treated mice (Shaw et al., 2000Go), with accompanying significant DNA strand breakage (Shen et al., 2002Go). A range of cytogenetic abnormalities have also been observed in human lymphoblastoid cells exposed to cylindrospermopsin (Humpage et al., 2000Go). The observed in vitro chromosomal effects suggested that cylindrospermopsin may cause damage to centromere/kinetochore function, although the mechanisms involved are yet to be determined.

Our previous studies on the toxicity of Aphanizomenon ovalisporum included the isolation of the toxic compounds, cylindrospermopsin and its isomer 7-epi cylindrospermopsin (Banker et al., 1997Go, 2000Go). The isolation of the nontoxic chlorine oxidation derivates of cylindrospermopsin, 5-chloro cylindrospermopsin, and cylindrospermic acid (Banker et al., 2001Go), suggested that the uracil moiety is crucial for the toxicity of cylindrospermopsin. In a recent study Runnegar et al. (2002)Go demonstrate that the sulfate group in the cylindrospermopsin structure did not play any role in the toxic activity or in the toxin uptake into the cells. Similar conclusion was obtained for the orientation of the hydroxyl group at the epimeric center C7. These structural/functional studies support the assumption (Banker et al., 2000Go) that the toxicity of cylindrospermopsin could stem from competitive inhibitory binding of the toxin to a catalytic site(s) involved in the synthesis of pyrimidine nucleotides (i.e., uridine). In the present study we estimated the effect of cylindrospermopsin on the in vitro activity of uridine monophospahte (UMP) synthase complex in a cell free liver extract from mice. We further evaluated the physiological responses of such inhibition in mice, assuming that cylindrospermopsin provided in drinking water might affect the two-step enzymatic conversion of oratate to UMP and lead to a metabolic disorder known as "orotic aciduria" with typical syndromes such as high level of orotic acid in the urine, retarded growth, and severe anemia caused by hypochromic erythrocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Weaned four-week-old male ICR mice (ca. 20 g, each) were supplied with food and water ad libitum for an experimental period of three weeks. Water containing cylindrospermopsin (0.6 mg/l equivalent to 1.44 µM) was provided to the experimental group (eight mice) whereas plain water was served to the control group (eight mice). The daily consumption of cylindrospermopsin was preplanned for the range of the no-observed-adverse-effect level (NOAEL) previously reported by Shaw et al. (2000)Go. Animals were maintained under standard conditions (21°C and a light dark cycle of 12 h) at the animal husbandry facilities of the Food Engineering and Biotechnology Faculty, Technion, Israel Institute of Technology, complying with the regulations of the local Ethics Committee for experimental studies in animals. Individual body weight was measured once a week. Water consumption and urine excretion were estimated once a week using metabolic cages, and urine samples were collected. The concentration of oratate in the urine samples was monitored by HPLC. Once a week a blood sample was drawn from the tail and hematocrit was determined. Red blood cells (RBC) and plasma were collected for the determination of total cholesterol content. At the end of the three weeks experimental period, animals were sacrificed by CO2 asphyxiation, the liver was removed, weighted, homogenized and sub-sampled for determination of total cholesterol level. Soluble crude protein extract was frozen and kept at –80°C until the performance of the in vitro UMP synthase assay.

Isolation and purification of cylindrospermopsin. Purified cylindrospermopsin was prepared from aqueous methanolic extraction of freeze dried culture of Aphanizomenon ovalisporum. The studied strain was isolated from Lake Kinneret during a 1994 bloom (Hadas et al., 1999Go; Pollingher et al., 1998Go). Cultures were grown batch wise in 20 l carboys containing BG11 medium pH 7.8 at 24 ± 1°C as previously described (Banker et al., 1997Go). The purification process of cylindrospermopsin included a flash chromatography on ODS-YMC Gel and final purification on an YMC ODS-AQ preparative HPLC column as was described by Banker et al. (2000)Go. Purified cylindrospermopsin was added to tap water to a final concentration of 0.6 mg/l (1.44 µM) and provided to mice ad libitum. Fresh dilution was daily prepared and the cylindrospermopsin concentration was periodically verified by analytical HPLC procedure using C18 column (YMC-Pack ODS-AQ 250 x 4.6 mm, 5 µm) maintained at 40°C. The chromatographic separation system was based on a mobile phase gradient from water (100%) to 5% methanolic aqueous solution. Elution solvents contained 5 mM phosphate buffer at pH 7.5. A UV detector set at 263 nm was used to identify the cylindrospermopsin peak which was quantified based on calibration curve of an authentic standard.

Fresh liver homogenate. Protein extract of fresh liver was prepared according to Raisonnier et al. (1981)Go. Untreated male ICR mice, weighing 25 g, were anesthetized and the liver was rapidly removed, washed in 0.15 M NaCl and homogenized using a Teflon homogenizer, in 10 ml/g of 20 mM This-HCl buffer (pH 7.4) containing 100 mM KCl, 5 mM MgCl2, 5 mM 5-phospho-D-ribosyl-1-pyrophosphate (PRPP) and 0.25 M sucrose. The homogenate was centrifuged for 20 min at 10,000 x g at 4°C, the supernatant was recovered and submitted for a second step of centrifugation for 10 min at 14,000 x g and 4°C to remove cells debris. Protein content in the supernatant was analyzed according to Bradford (1976)Go. The fresh liver homogenate was analyzed for the in vitro activity of UMP synthase complex.

UMP synthase activity in liver homogenate. The in vitro activity of UMP synthase complex was measured in fresh hepatic crude protein extract according to Krungkrai et al. (2001)Go. The assay volume mixture (200 µl) contained 0.1 mM orotic acid, 1 mM MgCl2, 0.5 mM 5-phosphoribosyl-1-pyrophosphate, 1 mM dithiotheritol, and 50 mM Tris-HCl pH 8. After incubation (without the fresh liver homogenate) at 37°C for 2 min, the reaction was initiated by the addition of fresh liver homogenate (total of 2.5 mg protein) and the mixture was incubated in a shaking water bath at 37°C for periods varied between 1.5 to 60 min. The reaction was terminated by the addition of 440 µl ice-cold HClO4 (1 M). The tubes were chilled on ice for 30 min, the denaturized protein was than removed by centrifugation for 10 min at 14,000 x g and the supernatant was collected and analyzed for the content of UMP by reversed-phase HPLC.

Kinetic properties of UMP synthase complex and inhibition by cylindrospermopsin. The kinetic parameters (Km and Vmax) of the UMP synthase complex in the hepatic crude protein extract were estimated from the reaction velocity response to various oratate concentrations. The basic in vitro assay was performed with different substrate concentrations varied between 0.5 and 25 µM orotic acid in the absence or the presence of cylindrospermopsin. Three concentrations of cylindrospermopsin (0.7, 2.7, and 4.7 µM) were examined in order to define the mode of inhibition.

Determination of cholesterol. Liver sample or red blood cell membranes were saponified in 0.3 ml of 40% KOH and 3 ml of 95% ethanol for 60 min at 80°C. Lipids were then extracted by petroleum ether (60–80°C). Cholesterol level was measured according to Seary and Bergquist (1960)Go using a calibration curve based on cholesterol solution in chloroform ranged between 0 and 0.4 mg/ml. The levels of total cholesterol in the plasma were assayed colorimetrically by using a commercial analytical kit (No. 402) from Sigma (St.Louis, MO).

Determination of orotic acid and UMP. The chromatographic separations were performed using reversed-phase LC-18 HPLC columns (250 x 4.6 mm i.d.; particle size 5 µm, Sigma). Orotic acid and UMP were separated using isocratic elution in 10 mM KH2PO4/H3PO4 buffer, pH 4, containing 10 mM KCl at a flow rate of 1 ml/min. The amount of orotic acid and UMP were calculated by simultaneously measuring their absorbance at 279 and 260 nm, respectively, and by comparing the peaks heights and areas with those of external standard and a calibration curve.

Microscopic analysis. For microscopic analysis of red blood cells (RBC) sample, 50 µl of blood was drawn from the mice tail and mixed with 50 µl of heparin 1000 unit/ml. RBC were collected by centrifugation at 3000 x g for 15 min. The supernatant was discard and 4 µl of the RBC were resuspended in 96 µl of PBS (pH 7.4) and observed by Nomarski light microscope. RBC were also prepared for examination by scanning electron microscopy (SEM). Aliquot of the RBC suspension was chemically fixed with an equal volume of glutardialdehyde (3%, v/v) for 3 min at room temperature. The suspension was then centrifuged at 6000 rpm for 3 min, and the supernatant discarded. The fixed cells were washed twice with distilled water and finally diluted with water. One drop from the final suspension was placed on a glass slide and allowed to dry in the air, at room temperature. The specimens were than covered with gold (~50 nm in thickness) by spatter coating and checked by SEM at acceleration voltage of 10 keV.

Statistical analysis. Statistical analyses were performed using SAS/Stat software (SAS Institute, Cary, NC).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kinetic Properties of UMP Synthase in Fresh Liver—The Effect of Cylindrospermopsin on in Vitro Activity
The activity of the UMP synthase complex was analyzed in a fresh hepatic crude protein extract of mice. A decrease in the orotic acid concentration concomitant with an increase in the UMP concentration was recorded during the incubation of the fresh liver extract with 0.1 mM oratate under standard assay conditions (Fig. 1). The reaction velocity calculated from the linear portion of the curve was 2.8 µmol UMP/mg protein/min. Running this assay with different initial concentrations of oratate showed a typical response of the reaction velocity to initial substrate concentrations, which could be well described by the Michaelis Menten kinetics (Fig. 2). The kinetics of the reaction converting orotate to UMP was affected by the presence of cylindrospermopsin as demonstrated in Figure 2. The Michaelis kinetic's parameter, Km, and the reaction maximal velocity, Vmax, were calculated by linearization of the experimental results presented in Figure 2 using Hanes-Woolf transformation (Fig. 3A). The calculated values are presented in Table 1 showing a clear effect of cylindrospermopsin on Vmax, which gradually decreased with the increasing of cylindrospermopsin concentration. While cylindrospermopsin at 0.7 µM imposed a statistically insignificant reduction of Vmax (p > 0.05), higher concentration of cylindrospermopsin led to a significant decrease in Vmax value from 3200 pmol UMP/mg protein/min in the absence of cylindrospermopsin, to 2450 and 2010 pmol UMP/mg protein/min, in the presence of 2.6 and 4.6 µM cylindrospermopsin, respectively (Table 1). No effect on Michaelis constant (Km) was observed with cylindrospermopsin concentration of 0.7 µM, whereas higher cylindrospermopsin concentrations only slightly, insignificantly, affected Km, from 1.36 µM oratate, in the absence of cylindrospermopsin, to 1.56 and 1.61 µM oratate in the presence of 2.6 and 4.6 µM cylindrospermopsin, respectively (Table 1). Such changes in the kinetic parameters indicate that cylindrospermopsin above a certain concentration (0.7 µM) affected mainly the reaction maximal velocity while the Michaelis constant (Km) was unaffected, thus the observed inhibition of UMP synthase complex by cylindrospermopsin could be characterized as a noncompetitive inhibition. The inhibition of the UMP synthase complex by cylindrospermopsin was further defined by an inhibition constant KI of 10 µM as extracted from plots of [S]/v versus I at varying substrate concentrations (Fig. 3B).



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FIG. 1. The in vitro transformation of Orotic acid to UMP by the UMP synthase complex in a crude protein extract from mice liver.

 


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FIG. 2. The effect of cylindrospermopsin on the in vitro activity of UMP synthase complex measured in protein extract from mice liver. Data presented are rates of the enzymatic transformation of orotic acid to UMP as function of substrate concentration. •–Control, without cylindrospermopsin, {circ}–0.7 µM cylindrospermopsin, {blacksquare}–2.6 µM cylindrospermopsin, {triangleup}–4.6 µM cylindrospermopsin. The experimental data was fitted to the Michealis Menten kinetic v = Vmax S/(Km + S).

 


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FIG. 3. Presentation of plots of [S]/v versus [S] (A) and [S]/v versus [I] (B) for the in vitro activity of UMP synthase complex in fresh protein extract from mice liver and the response to different cylindrospermopsin concentrations. (A) Data shown in Figure 2 was linearly transformed according to Hanes-Woolf equation, •–Control, without cylindrospermopsin, {circ}–0.7 µM cylindrospermopsin, {square}–2.6 µM cylindrospermopsin, {triangleup}–4.6 µM cylindrospermopsin. (B) [S]/v versus [I] relationships at various substrate concentration. The substrate concentrations are: •–2.4 µM orotic acid, {circ}–7.0 µM orotic acid, {blacktriangledown}–24.0 µM orotic acid.

 

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TABLE 1 Km and Vmax for the Two-Step Enzymatic Conversion of Orotic Acid to UMP

 
The Effect of Cylindrospermopsin on Metabolic Activities Related to the UMP Synthase Complex and Other Physiological Parameters
A group of eight mice was exposed to cylindrospermopsin (0.6 mg/l) in their drinking water for three weeks. Based on their daily water consumption (averaged at 2.8 ml/day), the daily cylindrospermopsin intake was 66 µg/kg BW. A control group of eight mice was provided with fresh untreated water ad libitum. A similar 25% gain in body weight was observed during the first and the second weeks both in the experimental and control groups. Body weight stabilized during the third week at about 32 g (Table 2). Following the three weeks exposure to cylindrospermopsin, the relative weights of liver, testes, and kidneys, in the experimental group, were higher compared with the control group (Fig. 4). No apparent difference was observed between the spleen relative weights of the exposed mice and the control (Fig. 4). Determination of the in vitro activity of UMP synthase complex in crude liver protein extract at the end of the experiment, indicated only a minor insignificant reduction in reaction rate (Vmax), measured at oratate saturating conditions, of the treated animals relative to the control group (data not shown).


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TABLE 2 Changes in Various Physiological Parameters Induced by Exposure of Mice to Cylindrospermopsin via Drinking Water

 


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FIG. 4. The relative weight of organs (g/100 g body weight) of mice exposed to cylindrospermopsin (0.6 mg/l) in their drinking water for three weeks (gray bar) in comparison with control group (black bar). An asterisk (*) indicate experimental results that are significantly different (p < 0.05) from the control.

 
Other metabolic parameters measured during the exposure period were urine excretion rate, oratate concentration in urine and blood hematocrit (Table 2). Daily urine excretion was gradually reduced in the cylindrospermopsin exposed group from 6.5 ± 1.1 at the beginning of the experiment to 3.9 ± 0.9 ml/100 g BW, after 21 days. Simultaneously, a significant increase in the urine orotic acid concentration was observed, from 0.20 ± 0.02 mM in the first week to 0.39 ± 0.04 and 0.40 ± 0.01 mM after 14 and 21 days, respectively (Table 2). In the control group the daily urine excretion was only slightly reduced after three weeks to 5.2 ± 1.8 ml/100 g BW, with only minor changes in the urine oratate concentration (Table 2). The daily excretion of oratate remained unaffected by the exposure to cylindrospermopsin (Table 2). Normal hematocrit level (46%) was observed in the control animals during the three-week experimental period. However, in mice exposed to cylindrospermopsin, the hematocrit gradually increased and reached a level of 52.1% after 21 days (Table 2).

The observed changes in the hematocrit were further studied by Nomarski light microscopy and SEM. These observations revealed substantial modifications in the outer shape of RBC in the cylindrospermopsin treated mice (Fig. 5). Minor morphological changes in the RBC were initially observed after the first week of exposure, and were gradually developed into an acanthocyte-like form, characterized by multiple spiny cytoplasmic irregularly spaced projections varied in width; usually contain a rounded end (Fig. 5).



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FIG. 5. Numarski light micrographs (A–D) and scanning electron micrographs (E and F) of red blood cells from mice exposed to cylindrospermopsin and from a control group (D and F). RBC were examined after one week (A), two weeks (B), and three weeks (C) of exposure ad libidum to cylindrospermopsin. Note the acanthocyte-like cells developed in the cylindrospermopsin exposed mice (marked with an arrow).

 
Liver and blood samples collected upon the termination of the experiment were analyzed for the abundance of cholesterol. The results presented in Figure 6 indicated that exposure to cylindrospermopsin imposed an increase in the cholesterol level in the RBC membranes and in the plasma, concomitant with a reduction in cholesterol level in the liver relative to the control group (Fig. 6).



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FIG. 6. Cholesterol content in red blood cell (RBC), plasma and liver of mice expose to cylindrospermopsin (0.6 mg/l) in drinking water for three weeks (gray bar) and in control group (black bar). The differences between the experimental and the control group are statistically significant (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on the structural modification of cylindrospermopsin and the toxicity of its different derivatives (Banker et al., 2001Go) we have suggested that the uracil moiety of cylindrospermopsin is crucial for the toxicity of the molecule. It was suggested that due to the uracil moiety the toxin can competitively bind to catalytic site/s involved in the synthesis and transformation of pyrimidine nucleotides (i.e., uridine). In the present study we assessed the inhibitory effect of cylindrospermopsin on the two sequential steps pathway that converts orotic acid to UMP via orotidine monophosphate. Two enzymes are involved in that pathway, orotic acid phosphoribosyl transferase (OPRT, EC 2.4.2.10) and orotidine 5-monophosphate decarboxylase (ODC, EC 4.1.1.23). The effect of cylindrospermopsin on the in vitro activity of UMP synthase complex in fresh liver homogenates was studied to determine the mode of inhibition. It was demonstrated that cylindrospermopsin significantly inhibited the enzymatic formation of UMP at concentrations above 0.7 µM. The inhibition was typed as a noncompetitive inhibition since the overall reaction maximal velocity, Vmax, was reduced, while the combined Michaelis constant, Km, was unaffected as the concentration of cylindrospermopsin increased. Mechanistically such an inhibition occurs when the inhibitor binds both to the free enzyme and the intermediate with the same equilibrium. However, since the in vitro reaction was based on two sequential reactions the precise mode of inhibition and the binding nature of cylindrospermopsin to either OPRT or ODC are yet unclear. An inhibition constant, KI, of 10 µM was calculated for the inhibition of UMP synthase complex by cylindrospermopsin. This suggests that the UMP synthase complex has a relatively low affinity to the toxin as compared with a Km value of 1.5 µM for orotic acid.

Three weeks exposure of mice to cylindrospermopsin imposed only a minor reduction in the maximal reaction rate, Vmax, of the in vitro activity of UMP synthase complex in crude liver protein extract. This observation could be attributed to the application of a relatively low dose (0.6 mg/l). Such a dose is slightly below the range of NOAEL reported by Shaw et al. (2000)Go. Assuming a preferential accumulation of cylindrospermopsin in the liver and a limited degradation of the toxin, the potential concentration of cylindrospermopsin in the liver could exceeded 40 µM (daily intake times 21 days divided by organ volume), i.e., four times greater than that of the KI calculated value for the inhibition of UMP synthase in vitro. While no data regarding distribution of cylindrospermopsin in mammalian tissues and organs were published yet, it is clear that these assumption are highly inaccurate In crayfish only portions of the uptaken cylindrospermopsin were concentrated in the hepatopancreas tissue and muscles, and in rainbow fish the toxin was evident only in the visceral tissues (Saker and Eaglesham, 1999Go). Furthermore, one cannot rule out xenobiotic degradation of cylindrospermopsin by hepatic cytochrome P450 (Shaw et al., 2000Go), as well as the renal filtration and excretion of the toxin in the urine. Therefore it is proposed that the toxin was concentrated in the liver to a level much lower than the KI, or alternatively washed away during the crude enzyme preparation. The minor reduction in the maximal reaction rate Vmax of UMP synthase further indicated that the application of cylindrospermopsin did not impose a down regulation of this enzymatic pathway.

The lack of a significant effect on the maximal reaction rate, Vmax, of the in vitro activity of UMP synthase complex in crude liver protein extract of the exposed mice, and the minor elevation of the amount of orotic acid excreted by the experimental animals rejected the initial working hypothesis that cylindrospermopsin might affect the enzymatic conversion of oratate to UMP and lead to a metabolic disorder known as "orotic aciduria." This disorder is characterized by a high level of orotic acid in the urine, retarded growth, and severe anemia caused by hypochromic erythrocytes (Milner and Visek, 1973Go). Nevertheless, the cylindrospermopsin-exposed mice developed other anomalous metabolic responses and pathological symptoms. The most striking effect of cylindrospermopsin in the in vivo experimental setup was the development of abnormal RBC formation known as acanthocytes, which was associated with a significant increase in the hematocrit. Studies on human and rats indicated that acanthocytes formed due to alterations in RBC membrane lipid content secondary to changes in plasma lipoproteins and a decrease in the activity of lecithin-acyl cholesterol transferase (LCAT), the enzyme that regulate the transfer of cholesterol between the RBC membrane and plasma (Gallagher and Forget, 2001Go; McBride and Jacob, 1970Go; Ulibarrena et al., 1994Go). These abnormalities lead to a marked increase in the ratio between free cholesterol and phospholipids in the RBC membrane (Williams et al., 1990Go). Mature erythrocytes lack the ability to de novo synthesize lipid components, and thus rely on lipids exchange with plasma lipoproteins and fatty acid acylation for phospholipids repair and renewal. LCAT in plasma catalyses the unidirectional pathway that depletes the erythrocytes membrane of cholesterol. This process is reversed in the absence of LCAT, or by its inactivation, resulting in a net accumulation of free cholesterol in the RBC membranes (Hochgraf et al., 1997Go). While direct evidences for the involvement of LCAT in the physiological responses to cylindrospermopsin are missing, it was reported that severe liver or renal failures inactivate LCAT (Cooper and Jandl, 1968Go, Gillet et al., 2001Go). Falconer et al. (1999)Go demonstrated that cylindrospermopsin administration caused both liver and renal damage in mice. Thus, an indirect effect of cylindrospermopsin on plasma LCAT activity can not be ruled out.

Furthermore, the observed variation in cholesterol levels in the plasma and liver of cylindrospermopsin-exposed mice might also be attributed to a direct or indirect effect of cylindrospermopsin on cholesterol metabolism via oxidative state. There are two pathways in cholesterol metabolism that could be affected (Staprans et al., 1993Go): (1) Oxidized lipoproteins hardly hydrolyzed by lipoprotein lipase and therefore impair cholesterol uptake by the liver and (2) The amount of oxidized lipids in chylomicrons was inversely correlated with reduced glutathione (GSH) content of the intestinal mucosa cells. Taking into account that cylindrospermopsin imposed reduction of GSH content in liver (Runnegar et al., 1994Go, 1995Go, 2002Go), it is proposed that the hepatic uptake of lipoproteins is affected by cylindrospermopsin via alteration of oxidation-mediated cholesterol metabolism.


    NOTES
 

1 To whom correspondence should be addressed. Fax: 972-4-6724627. E-mail: assaf{at}ocean.org.il.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Banker, R., Carmeli, S., Hadas, O., Teltsch, B., Porat, R., and Sukenik, A. (1997). Identification of cylindrospermopsin in the cyanobacterium Aphanizomenon ovalisporum (Cyanophyceae) isolated from Lake Kinneret, Israel. J. Phycol. 33, 613–616.[ISI]

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Hadas, O., Pinkas, R., Delphin, E., Vardi, A., Kaplan, A., and Sukenik, A. (1999). Limnological and ecophysical aspects of Aphanizomenon ovalisporum bloom in Lake Kinneret, Israel. J. Plankton Res. 21, 1439–1453.[Abstract]

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