* Department of Food Engineering and Biotechnology, Technion, Israel Institute of Technology, Haifa 32000 Israel; School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel;
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
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ABSTRACT |
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Key Words: hepatotoxin; cyanobacteria; cylindrospermopsin; UMP synthase; cholesterol.
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INTRODUCTION |
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Cylindrospermopsin was shown to be a potent inhibitor of protein synthesis. Terao et al. (1994) 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. (1994
, 2002
) 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., 1995
). Norris et al. (2002)
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., 2000), with accompanying significant DNA strand breakage (Shen et al., 2002
). A range of cytogenetic abnormalities have also been observed in human lymphoblastoid cells exposed to cylindrospermopsin (Humpage et al., 2000
). 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., 1997, 2000
). The isolation of the nontoxic chlorine oxidation derivates of cylindrospermopsin, 5-chloro cylindrospermopsin, and cylindrospermic acid (Banker et al., 2001
), suggested that the uracil moiety is crucial for the toxicity of cylindrospermopsin. In a recent study Runnegar et al. (2002)
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., 2000
) 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.
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MATERIALS AND METHODS |
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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., 1999; Pollingher et al., 1998
). 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., 1997
). 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)
. 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). 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)
. 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). 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 (6080°C). Cholesterol level was measured according to Seary and Bergquist (1960) 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).
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RESULTS |
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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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DISCUSSION |
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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). 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, 1999
). Furthermore, one cannot rule out xenobiotic degradation of cylindrospermopsin by hepatic cytochrome P450 (Shaw et al., 2000
), 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, 1973). 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, 2001
; McBride and Jacob, 1970
; Ulibarrena et al., 1994
). These abnormalities lead to a marked increase in the ratio between free cholesterol and phospholipids in the RBC membrane (Williams et al., 1990
). 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., 1997
). 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, 1968
, Gillet et al., 2001
). Falconer et al. (1999)
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., 1993): (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., 1994
, 1995
, 2002
), it is proposed that the hepatic uptake of lipoproteins is affected by cylindrospermopsin via alteration of oxidation-mediated cholesterol metabolism.
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NOTES |
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1 To whom correspondence should be addressed. Fax: 972-4-6724627. E-mail: assaf{at}ocean.org.il.
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