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Application of precolumn oxidation HPLC method with fluorescence detection to evaluate saxitoxin levels in discrete brain regions of rats
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Toxicon 49 (2007) 89–99 www.elsevier.com/locate/toxicon
Application of precolumn oxidation HPLC method with fluorescence detection to evaluate saxitoxin levels in discrete brain regions of rats Rosa Carmina Cervantes Ciancaa,b,, Miguel Alfonso Pallaresb, Rafael Dura´n Barbosab, Lucı´ a Vidal Adanb, J. Manuel Lea˜o Martinsc, Ana Gago-Martı´ nezc a Departamento de Ciencias Marinas, Instituto Tecnolo´gico de Boca del Rı´o, Ver. Me´xico Departamento de Biologı´a Funcional y Ciencias de la Salud, Facultad de Biologı´a, Campus Universitario de Vigo, Universidad de Vigo, 36200 Vigo, Spain c Departamento de Quı´mica Analı´tica y Alimentaria, Facultad de Quimica, Campus Universitario de Vigo, Universidad de Vigo, 36200 Vigo, Spain
b
Received 19 June 2006; accepted 20 September 2006 Available online 29 September 2006
Abstract Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel and for this reason, it has been widely used in the study of Na+ channel. The aim of this study was to analyze STX distribution in different rat brain regions after its acute intraperitoneal (i.p.) administration. Male rats (150–200 g) were injected i.p. with STX (5 and 10 mg/kg of body weight). After three time intervals of 30, 60, and 120 min (for 5 mg/kg STX dose) and 30 min (for 10 mg/kg STX dose) animals were sacrificed by cervical dislocation. Brains were removed and dissected in seven regions. STX concentration was measured using a precolumn oxidation high-performance liquid chromatographic method with fluorescence detection (HPLC/FLD). STX was found in all the regions evaluated at ppm levels meaning that STX peripherical administered across the blood–brain barrier and is distributed along the whole brain. r 2006 Elsevier Ltd. All rights reserved. Keywords: Saxitoxin; HPLC; Brain regions; Analytical method
1. Introduction Paralytic shellfish poisoning (PSP) toxins, in which saxitoxin (STX) is included, constitute a Corresponding author. Departamento de Biologı´ a Funcional y Ciencias de la Salud, Facultad de Biologı´ a, Campus Universitario de Vigo, Universidad de Vigo, 36200 Vigo, Spain. Tel.: +34 986 81 19 96; fax: +34 986 81 25 56. E-mail address: [email protected] (R.C.C. Cianca).
0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.09.021
group of highly toxic compounds, mainly produced by marine dinoflagellates during their growth period under favorable environmental conditions. Overgrowth of these organisms is linked to marine toxic events commonly known as ‘‘red tides’’ which are often associated with massive fish, bird, and marine animal kills (Davis, 1948). These events occur naturally, and their frequency, duration, and worldwide distribution have been considerably increased
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over the last few years, being associated with the eutrophication of marine and freshwater (Hallegraef, 1992; Hungerford, 2005). The main species responsible for this contamination in marine waters are three morphologically distinct genera of dinoflagellates (Alexandrium sp., Pyrodinium sp., and Gymnodinium sp.), while four different species of cyanobacteria or bluegreen algae (Anabaena circinalis, Aphanizomenon flosaquae, Cylindrospermopsis raciborkii, and Lyngbya wollei) are the main species responsible in freshwaters (Shimizu, 1978; Lizet de Leon, 2005). STX and analogues are powerful neurotoxic compounds. Their main toxicological activity is observed through the blockade of the sodium channels (Henderson et al., 1973; Strichartz, 1984; Guo et al., 1987; Hu and Kao, 1991). These neurotoxins can originate a reduction in the amplitude and speed of conduction of the action potentials by the peripheral and central nerves as well as a weakening of skeletal muscle contraction (Lipkind, 1994). A variety of marine neurotoxins exert potent and specific actions on neuronal sodium channels. Tetrodotoxin (TTX) and STX selectively block the sodium channel without any effect on other types of voltage-activated and transmitter-activated ion channels. They bind to a site near the external pore of the sodium channel on a one-to-one stoichiometric basis. The block is influenced by the membrane potential in a complex manner, while binding and entry of calcium ions to the sodium channel appear to be responsible for the voltagedependent blockade. Owing to the potent and specific sodium channel blocking action, TTX and STX have been used extensively in various studies of ion channels (Narahashi et al., 1994). There is, however, limited information on STX binding properties and neuroanatomical distribution of the sodium channels through the rat brain regions (Xia and Haddad, 1993). Nevertheless, even STX mechanism is well-known; there is no complete evidence if it can cross the blood–brain barrier and about its distribution in discrete brain regions. This is mainly due to technical problems relating to the method used to quantify STX in body fluids and tissues (Andrinolo et al., 2002). However, the optimum conditions for STX determination in tissues were not fully investigated and this work is the first application of the said method to evaluate STX in rat’s brain tissues. This evaluation was carried out by injecting male rats
with STX at doses of 5 and 10 mg/kg of body weight (bw) in order to evaluate the dynamics of STX distribution in different rat brain regions following acute intraperitoneal (i.p.) administration of STX. 2. Materials and methods 2.1. Toxin Standard solution of STX was purchased from the Institute for Marine Bioscience, National Research Council, Certified Reference Material Program (NRC-CRM) from Halifax, Canada. STX was diluted in 0.003 M hydrochloric acid (HCl) to a final concentration of 1.944 ng/ml. 2.2. Animals Male adult Sprague–Dawley rats (weighing between 150 and 200 g) were used in all the experiments. Animals were housed under monitored conditions of temperature (2272 1C) and photoperiod (light:dark 14:10 h) with free access to food and water. The experiments were performed according to the guidelines of the European Union Council (86/609/EU) for the use of laboratory animals (Guidelines, 1986). 2.3. Reagents All solvents and chemical reagents were highperformance liquid chromatographic (HPLC) or analytical grade. Hydrogen peroxide, acetic acid, sodium hydroxide (NaOH), HCl, perchloric acid (HClO4), and acetonitrile were purchased from Panreac. Ammonium format was purchased from sigma and the water used was obtained from a Milli-Q System (Millipore Ltd.). 2.4. Determination of STX in brain regions We studied two doses of STX (5 and 10 mg/kg bw) in groups of 18 rats. Lower dose (approximately 0.5 ml suspension of STX) was directly injected (i.p.) using a syringe, amounting to the equivalent of 5 mg/ kg bw. Higher dose (approximately 1 ml suspension of STX) was also injected directly (i.p.), amounting to the equivalent of 10 mg/kg bw. After systemic administration, we just wait different time periods in order to know STX levels that reach to the rat brain, so that animals were sacrificed by cervical dislocation at the ending of each one. Time periods
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were 30, 60, and 120 min for lower dose and 30 min for the higher dose. As soon as rats were killed, brains were removed (three brain samples put together and homogenized as one sample in each case) and dissected in the following rat brain regions: hypothalamus (H), striatum (S), brain stem (BS), midbrain (MB), frontal cortex (FC), left hemisphere (HL), and right hemisphere (HR). Three control rats were killed in parallel to both treated groups post-injection of 0.5–1.0 ml of saline solution. 2.5. Sample preparation Tissue samples were weighed and then homogenized by sonication in a solution of 0.1 M HClO4 (H, S, BS, MB, and FC in a proportion of 1:20; and LH and RH in a proportion of 1:8), the pH being adjusted to 3–4 with NaOH 0.1 M, and then boiled to 60 1C for 5 min. After that, the samples were centrifuged at 16,000g (4 1C) for 15 min. Supernatants were filtered through 0.22 mm nylon filters, concentrated in a rotovapor STUART RE300 until dryness, and then redissolved in 0.003 M HCl. Redissolved samples were centrifuged in ultrafreeMC Millipore (Waters) at 5000g during 5 min. About 300 ml of the filtrate was applied to hydrogen peroxide oxidation, as describe by Lawrence et al. (1995) for STX determination by HPLC/fluorescence detection (FLD). 2.6. Determination of STX by HPLC/FLD This method was based on the hydrogen peroxide oxidation of STX described by Lawrence et al. (1995) and modified as follows: 250 ml of 1 M NaOH and 25 ml of 3% hydrogen peroxide were added to 300 ml of the standard solution or the sample obtained after extraction procedure. The mixture was allowed to react for 2 min at room temperature. About 25 ml of 99.7% acetic acid was added to stop the reaction. Finally, 20 ml of this solution was injected into the HPLC system. 2.7. Chromatographic conditions for HPLC analysis The STX was analyzed under the conditions described previously using hydrogen peroxide oxidation as described by Lawrence et al. (1995) with slight modifications. Briefly, a 20 ml pretreated sample was injected into the HPLC system using a Rheodine 7125 injection valve. The separation of
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STX was achieved using a Phenomenex Luna C18 100P reversed-phase column (250 4.6 mm2 and 5 mm particle size). The HPLC–FLD system was an Agilent System 1100, equipped with a Quaternary HPLC pump, mobile-phase degasification system and fluorescence detector. Data were analyzed by the Agilent HP chemstation software. The column was eluted with a gradient program of solvents consisting of 0–100% (v/v) acetonitrile in 0.1 M ammonium format, adjusted to pH 6 with 0.1 M HCl, as follows: 0–5% during the first 5 min, then 5–70% during the next 4 min, then 70–100% during the next 9 min, and finally, 100–0% during the last 2 min. The flow rate was maintained at 1.5 ml/min. STX was detected with a fluorescence detector with an excitation wavelength of 330 nm and an emission wavelength of 390 nm. 2.8. Calibration studies To show linearity, a calibration curve in the range of 0.05–20 ng/ml STX was obtained. Detection and quantification limits have been evaluated on the basis of signal/noise 3:1 and 10:1, respectively. Reproducibility studies were carried out by injecting a 16 ng/ml STX standard solution into the HPLC/ FLD system, parameters such as: mean, standard deviation (SD), standard error of the mean (SEM), and variability coefficient in the area peaks were also evaluated. The HPLC/FLD method once optimized was applied for the determination of STX in the samples corresponding to different regions of the rat’s brain evaluated. 2.9. STX recovery from brain samples Recovery experiments were carried out to evaluate the efficiency of the extraction method; three control brains (total brain was suspended in 10 ml of HClO4) were spiked with 40 ng/ml of STX standard. Also brain control regions were prepared, and each region was spiked with 16 ng/ml of STX standard. After 30 min, the sample pretreatment described above was applied and these studies were carried out in triplicate. 2.10. Statistical evaluation Data of STX levels in discrete brain regions are expressed by means7SEM. Statistical evaluation of
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Ar
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Fig. 1. (a) Time retention (tR ¼ 14.5) and the area of the peak of the standard after the injection of 20 ml of STX standard (16 ng/ml). (b) A chromatogram obtained with the 10 mg/kg dose group after sample preparation under the conditions depicted in Section 2.6.
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the results obtained was performed by using ANOVA followed by Student–Newman–Keuls test. 3. Results 3.1. Calibration studies Liquid chromatographic analysis with FLD was carried out under the conditions described. Fig. 1a shows time retention (tR ¼ 14.5) and the area of the peak of the standard. Fig. 1b shows a chromatogram obtained with the 10 mg/kg dose group after sample preparation. About 20 ml of the different concentration range was injected in triplicate in the HPLC system. Good linearity (R2 ¼ 0.9916) in the 0.05–20 ng/ml STX range concentration was observed (Fig. 2). Detection limit was 11.1970.11 STX pg and quantification limit was 32.4770.14 STX pg (20 ml injected respectively). Time retention (tR) and area obtained in the reproducibility of the method, tR was 14.1870.56 min, and the area peak was 2.0170.07 FLD u.a. (n ¼ 10), each sample (16 ng/ml) was injected in triplicate. 3.2. Determination of STX in the brain of rats After sample preparation, STX concentration present in the brain of the rats was measured. Due to the low levels of STX in the different samples evaluated, a preconcentration step has been required involving evaporating the samples until dryness, redissolving in 0.003 M HCl. By
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the way, increasing the sample extract volume from 100 to 300 ml (modification of Lawrence et al. (1995)), chromatographic detection was enhanced (Fig. 3). The oxidation reagent volume was not increased. Fig. 4 shows each of the regions of the brain that have been tested. 3.3. STX recovery from brain samples Relative recovery of samples spiked with 40 ng/ml of STX standard for total brain and 16 ng/ml for each one of the regions tested (% recovery7RSD with n ¼ 3) was 59.5%71.5 for total brain; 47.6%71 for H; 47.2%71 for S; 49.8%71.8 for BS; 49.5%71.9 for MB; 49.6%71.9 for FC; 52.1%72.5 for LH; and 52.3%72.5 for RH. The mean of all the samples was 49.72%71.8. The analysis of the results led us to conclude that the recovery percentage was better in the region with higher weight. Although recoveries were approximately 50%, they were considered to be acceptable to evaluate the concentration of STX in the different regions of the brain considering the complexity of the matrix employed. 3.4. STX levels in brain regions Figs. 5a–c show the STX concentration in H, S, BS, MB, FC, LH, and RH rat brain regions after systemic administration of 5 mg/kg, at observation times of 30, 60, and 120 min at conditions described
3.00 y = 0.1264x - 0.0732 R2 = 0.9916
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FLD1 A, Ex=330, Em=390 (STXRATAS\04040504.D) FLD1 A, Ex=330, Em=390 (STXRATAS\04040505.D) FLD1 A, Ex=330, Em=390 (STXRATAS\04040508.D)
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in Section 2.4. STX concentration varies from 0.7670.02 to 2.3670.19 pg/mg of H; from 0.57 0.02 to 1.1870.28 pg/mg of S; from 0.2670.01 to 1.5070.15 pg/mg of BS; from 0.2770.02 to 1.387 0.04 pg/mg of MB; 0.5870.03 to 1.5170.13 pg/mg of FC; from 0.3170.01 to 0.7670.15 pg/mg of LH, and from 0.3270.02 to 0.8070.2 pg/mg of RH, in each case the first valour corresponds to 30 min and the last one corresponds to 120 min time periods. It was observed that maximum concentration STX was found in H and minimum concentration was
observed in LH and RH. A proportional increase of STX level was also observed in each region studied across the reaction time. Fig. 5d shows the STX concentration in H, S, BS, MB, FC, LH, and RH rat brain regions at 30 min after systemic administration of 10 mg/kg. STX concentration was in H 3.570.22 pg/mg; S 3.67 0.02 pg/mg; BS 3.5370.02 pg/mg; MB 2.747 0.02 pg/mg; FC 3.4570.05 pg/mg; LH 1.7770.10 pg/mg, and RH 1.5670.07 pg/mg. Significance differences (Po0.05) to which regions were
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FC
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Fig. 4. Diagram of the brain identifying each of the regions of the brain that has been tested (FC ¼ Frontal cortex; He ¼ Hemispheres; S ¼ Striatum; H ¼ hypothalamus; MB ¼ Midbrain; BS ¼ Brain stem).
significantly different from one another are also shown in Figs. 5a–d. The total content of STX in the whole brain with the 5 mg/kg dose was 0.5370.016, 0.7570.11, and 1.3270.22 ng/ml at 30, 60, and 120 min, respectively. These values represent 7.12%, 10%, and 17.64% in each case. As regards the 10 mg/kg dose, the total content of STX was 3.6170.33 ng, which represents 24.06% in the whole brain. Fig. 6 shows the linearity tendency in the dose answer in the different brain regions studied over time after the systemic administration of 5 mg/kg. 4. Discussion To date, the main impediment for evaluating the toxic effects of STX was the lack of sensitivity and reproducibility for the detection of STX at pg levels. Xia and Haddad (1993) used a broad range of 3H–STX concentration (up to 64 nM) to examine saturation profiles and density distribution in adult rats. They found that STX sites do not vary greatly in affinity (most Kds ¼ 2–5 nM) in various regions of the adult rat brain; STX binding distribution was very heterogeneous in the rat with a much higher density in the cortex, hippocampus, amygdala, and cerebellum than in the brainstem and spinal cord; STX sites are mostly localized in layers mainly of neurons with low density in white matter. Recently, Andrinolo et al. (1999) developed an isolation procedure coupled with post-column derivatization HPLC method that was capable of
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detecting quantities as low as one pM of STX in body fluids and tissue samples of adults cat anaesthetized and intravenously injected with low (2.7 mg of STX/Kg) and high doses (10 mg of STX/ Kg) of toxin (Andrinolo et al., 1999). They proved that the technique developed by Oshima (1995) was quite powerful. Until now, there has been no report in the literature about a method that succeeds in quantifying STX or its analogues in different brain regions of rats, after systemic administration of the toxin. In our study, a successful evaluation of STX at ppm levels in different brain regions has been achieved using a precolumn oxidation HPLC-LD method with hydrogen peroxide as oxidizing reagent. Slight modifications on the method developed by Lawrence et al. (1995) have been included to obtain the optimal conditions for this particular matrix. Good sensitivity was obtained and a good reproducibility was observed. Nevertheless, the recovery extraction procedure about spiked brain samples tested with STX was about 50%. The research of STX levels that reach the rat brain has been complicated due to the relative complexity of the matrix employed, biological tissues (Hines et al., 1993; Andrinolo et al., 1999). As soon as rats were killed, brain samples were proceeded and analyzed; nevertheless, we detected no STX, so that we combined the brain of three animals, from 1 to 3, obtaining a good response on STX determination in the different brain regions analyzed. A substantial increase about time response can be observed in the different brain regions studied with low dose of STX, the increase was proportional to the time after STX administration. We found that STX distribution was heterogeneous, and observed STX levels in all the regions studied. After 30 min following administration of STX, the highest concentration was detected in H, S, and FC with lower STX concentration in the other regions. Conversely, at 60 and 120 min, H, and FC continue to the region with the highest concentration of toxin detected; nevertheless, in BS and MB, STX concentration was increased, but in S the increase was least less. Hemispheres always present lower concentration independently, regardless of the time interval studied. It is possible that the regions with the higher STX concentration are more accessible to the toxin since the periphery and/or they are formed in layers mostly made of neurons
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with low density in white matter (Xia and Haddad, 1993); over the time, STX is distributed to other regions.
The death of rats with high dose of STX, 10 mg/ kg, after 30 min can be produced by arrest shock place (Kao et al., 1967; Nagasawa et al., 1971;
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Fig. 5. (a) Concentration of STX in different rat brain regions after 30 min of i.p. administration (5 mg/Kg). Significance difference Po0.05 respects to 1S, BS, MB, FC, LH, RH; 2H, BS, MB,LH, RH; 3H, S, FC; 4H, S, FC; 5H, BS, MB, LH, RH; 6,7H, S, FC. The values represent the mean and SEM obtained from six determinations. (b) Concentration of STX in different rat brain regions after 60 min of i.p. administration (5 mg/Kg). Significance difference Po0.05 respects to 1S, BS, LH, RH; 2H, MB, FC, LH, RH; 3H, MB, FC; 4S, BS, FC, LH, RH; 5S, BS, LH, RH; 6,7H, S,MB, FC. The values represent the mean and SEM obtained from six determinations. (c) Concentration of STX in different rat brain regions after 120 min of i.p. administration (5 mg/Kg). Significance difference Po0.05 respects to 1S, BS, MB, FC, LH, RH; 2H; 3H, LH, RH; 4H, LH, RH; 5H, LH, RH; 6,7H, BS,MB, FC. The values represent the mean and SEM obtained from six determinations. (d) Concentration of STX in different rat brain regions after 30 min of i.p. administration (10 mg/Kg). Significance difference Po0.05 respects to 1MB, LH, RH; 2MB, LH, RH; 3MB, LH, RH; 4H, S, BS, FC, LH, RH; 5MB, LH, RH; 6,7H, S, BS,MB, FC. The values represent the mean and SEM obtained from six determinations.
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Borinson et al., 1980; Chang et al., 1992), so involves both central and peripheral cardio-respiratory system components (Andrinolo et al., 1999). After STX systemic administration, STX levels in the different brain regions studied are higher than STX levels at 120 min with 5 mg/kg dose, meaning that there is a faster distribution when STX concentration is higher (near to LD50). In this case, it is observed that almost all the regions have similar STX concentration, except the hemispheres.
The STX concentration found in total brain, with the two doses, showed that a part of STX administered i.p. can cross the blood–brain barrier and produce some alterations in the central nervous system, regardless of the effects produced in the periphery. The results obtained show that the precolumn oxidation method is suitable for such particular application providing fast and sensitive information about the distribution of STX in the different rat brain regions analyzed.
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RH Fig. 6. Results obtained in pg of STX for mg of tissue in the different brain regions studied, after i.p. injection of STX (5 mg/Kg b.w.) to male rat. After 30, 60, and 120 min. Tissues were taken from the animals, processed as detailed under Materials and Methods, and subjected to HPLC/FLD analysis for STX. The values represent the mean and SEM obtained from six determinations. (a) Po0.05 significance difference respects to 30. (b) Po0.05 significance difference respects to 60. (c) Po0.05 significance difference respects to 120.
Acknowledgments Financial support from the University of Vigo, the Regional Government of Galicia, and AbKem Iberia for supplying materials and reagents for the development of this work is acknowledged.
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ARTICLE IN PRESS R.C.C. Cianca et al. / Toxicon 49 (2007) 89–99 Hallegraef, G.M., 1992. A review of harmful algal blooms and their apparent global increase. Phycologia 32, 79–99. Henderson, R., Ritchie, J.M., Strichartz, G.R., 1973. The binding of labelled saxitoxin to the sodium channel in nerve membranas. J. Physiol. 235, 783–804. Hines, H.B., Naseem, S.M., Wannemacher Jr., R.W., 1993. 3HSaxitoxinol metabolism and elimination in the rat. Toxicon 31, 905–908. Hu, S.L., Kao, C.Y., 1991. Interactions of neosaxitoxin with the sodium channel of the frog skeletal muscle fiber. J. Gen. Physiol. 97, 561–578. Hungerford, J.M., 2005. Committee on natural toxins and food allergens marine and fresh water toxins. J. AOAC Int. 88, 229–313. Kao, C.Y., Suzuki, C.Y., Kleinahus, T., Siegman, M.J., 1967. Vasomotor and respiratory depressant actions of tetrodotoxin and saxitoxin. Arch. Int. Pharmacodyn. 165, 438–450. Lawrence, J.F., Me´nard, C., Cleroux, Ch., 1995. Evaluation of prechromatographic oxidation for liquid chromatography determination of paralytic shellfish poisons in shellfish. J. AOAC Int. 78, 514–520.
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Lipkind, G., 1994. A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel. Biophy. J. 66 (1), 1–13. Lizet de Leon, 2005. Floraciones algales de agua dulce. Cianobacterias, cianotoxinas, Animales ponzon˜osos y toxinas biolo´gicas. Oficina del Libro-FEFMUR, pp. 675–682. Nagasawa, J., Spiegelstein, M.Y., Kao, C.Y., 1971. Cardiovascular actions of saxitoxin. J. Pharmacol. Exp. Ther. 178, 103–109. Narahashi, T., Roy, M.L., Ginsburg, K.S., 1994. Recent advances in the study of mechanism of action of marine neurotoxins. Neurotoxicology 15 (3), 545–554. Oshima, Y., 1995. Post column derivatization liquid chromatographic method for paralytic shellfish toxins. J. AOAC Int. 78, 528–532. Shimizu, Y., 1978. Dinoglagellate toxins. In: Scheuer, P.J. (Ed.), Mar.Nat.Products, pp. 1–42. Strichartz, G., 1984. Structural determinants of the affinity of saxitoxin sodium channel. J. Gen. Physiol. 84, 281–305. Xia, Y., Haddad, G.G., 1993. Neuroanatomical distribution and binding properties of saxitoxin sites in the rat and turtle CNS. J. Comp. Neurol. 330 (3), 363–380.
Toxicon 49 (2007) 89–99 www.elsevier.com/locate/toxicon
Application of precolumn oxidation HPLC method with fluorescence detection to evaluate saxitoxin levels in discrete brain regions of rats Rosa Carmina Cervantes Ciancaa,b,, Miguel Alfonso Pallaresb, Rafael Dura´n Barbosab, Lucı´ a Vidal Adanb, J. Manuel Lea˜o Martinsc, Ana Gago-Martı´ nezc a Departamento de Ciencias Marinas, Instituto Tecnolo´gico de Boca del Rı´o, Ver. Me´xico Departamento de Biologı´a Funcional y Ciencias de la Salud, Facultad de Biologı´a, Campus Universitario de Vigo, Universidad de Vigo, 36200 Vigo, Spain c Departamento de Quı´mica Analı´tica y Alimentaria, Facultad de Quimica, Campus Universitario de Vigo, Universidad de Vigo, 36200 Vigo, Spain
b
Received 19 June 2006; accepted 20 September 2006 Available online 29 September 2006
Abstract Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel and for this reason, it has been widely used in the study of Na+ channel. The aim of this study was to analyze STX distribution in different rat brain regions after its acute intraperitoneal (i.p.) administration. Male rats (150–200 g) were injected i.p. with STX (5 and 10 mg/kg of body weight). After three time intervals of 30, 60, and 120 min (for 5 mg/kg STX dose) and 30 min (for 10 mg/kg STX dose) animals were sacrificed by cervical dislocation. Brains were removed and dissected in seven regions. STX concentration was measured using a precolumn oxidation high-performance liquid chromatographic method with fluorescence detection (HPLC/FLD). STX was found in all the regions evaluated at ppm levels meaning that STX peripherical administered across the blood–brain barrier and is distributed along the whole brain. r 2006 Elsevier Ltd. All rights reserved. Keywords: Saxitoxin; HPLC; Brain regions; Analytical method
1. Introduction Paralytic shellfish poisoning (PSP) toxins, in which saxitoxin (STX) is included, constitute a Corresponding author. Departamento de Biologı´ a Funcional y Ciencias de la Salud, Facultad de Biologı´ a, Campus Universitario de Vigo, Universidad de Vigo, 36200 Vigo, Spain. Tel.: +34 986 81 19 96; fax: +34 986 81 25 56. E-mail address: [email protected] (R.C.C. Cianca).
0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.09.021
group of highly toxic compounds, mainly produced by marine dinoflagellates during their growth period under favorable environmental conditions. Overgrowth of these organisms is linked to marine toxic events commonly known as ‘‘red tides’’ which are often associated with massive fish, bird, and marine animal kills (Davis, 1948). These events occur naturally, and their frequency, duration, and worldwide distribution have been considerably increased
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over the last few years, being associated with the eutrophication of marine and freshwater (Hallegraef, 1992; Hungerford, 2005). The main species responsible for this contamination in marine waters are three morphologically distinct genera of dinoflagellates (Alexandrium sp., Pyrodinium sp., and Gymnodinium sp.), while four different species of cyanobacteria or bluegreen algae (Anabaena circinalis, Aphanizomenon flosaquae, Cylindrospermopsis raciborkii, and Lyngbya wollei) are the main species responsible in freshwaters (Shimizu, 1978; Lizet de Leon, 2005). STX and analogues are powerful neurotoxic compounds. Their main toxicological activity is observed through the blockade of the sodium channels (Henderson et al., 1973; Strichartz, 1984; Guo et al., 1987; Hu and Kao, 1991). These neurotoxins can originate a reduction in the amplitude and speed of conduction of the action potentials by the peripheral and central nerves as well as a weakening of skeletal muscle contraction (Lipkind, 1994). A variety of marine neurotoxins exert potent and specific actions on neuronal sodium channels. Tetrodotoxin (TTX) and STX selectively block the sodium channel without any effect on other types of voltage-activated and transmitter-activated ion channels. They bind to a site near the external pore of the sodium channel on a one-to-one stoichiometric basis. The block is influenced by the membrane potential in a complex manner, while binding and entry of calcium ions to the sodium channel appear to be responsible for the voltagedependent blockade. Owing to the potent and specific sodium channel blocking action, TTX and STX have been used extensively in various studies of ion channels (Narahashi et al., 1994). There is, however, limited information on STX binding properties and neuroanatomical distribution of the sodium channels through the rat brain regions (Xia and Haddad, 1993). Nevertheless, even STX mechanism is well-known; there is no complete evidence if it can cross the blood–brain barrier and about its distribution in discrete brain regions. This is mainly due to technical problems relating to the method used to quantify STX in body fluids and tissues (Andrinolo et al., 2002). However, the optimum conditions for STX determination in tissues were not fully investigated and this work is the first application of the said method to evaluate STX in rat’s brain tissues. This evaluation was carried out by injecting male rats
with STX at doses of 5 and 10 mg/kg of body weight (bw) in order to evaluate the dynamics of STX distribution in different rat brain regions following acute intraperitoneal (i.p.) administration of STX. 2. Materials and methods 2.1. Toxin Standard solution of STX was purchased from the Institute for Marine Bioscience, National Research Council, Certified Reference Material Program (NRC-CRM) from Halifax, Canada. STX was diluted in 0.003 M hydrochloric acid (HCl) to a final concentration of 1.944 ng/ml. 2.2. Animals Male adult Sprague–Dawley rats (weighing between 150 and 200 g) were used in all the experiments. Animals were housed under monitored conditions of temperature (2272 1C) and photoperiod (light:dark 14:10 h) with free access to food and water. The experiments were performed according to the guidelines of the European Union Council (86/609/EU) for the use of laboratory animals (Guidelines, 1986). 2.3. Reagents All solvents and chemical reagents were highperformance liquid chromatographic (HPLC) or analytical grade. Hydrogen peroxide, acetic acid, sodium hydroxide (NaOH), HCl, perchloric acid (HClO4), and acetonitrile were purchased from Panreac. Ammonium format was purchased from sigma and the water used was obtained from a Milli-Q System (Millipore Ltd.). 2.4. Determination of STX in brain regions We studied two doses of STX (5 and 10 mg/kg bw) in groups of 18 rats. Lower dose (approximately 0.5 ml suspension of STX) was directly injected (i.p.) using a syringe, amounting to the equivalent of 5 mg/ kg bw. Higher dose (approximately 1 ml suspension of STX) was also injected directly (i.p.), amounting to the equivalent of 10 mg/kg bw. After systemic administration, we just wait different time periods in order to know STX levels that reach to the rat brain, so that animals were sacrificed by cervical dislocation at the ending of each one. Time periods
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were 30, 60, and 120 min for lower dose and 30 min for the higher dose. As soon as rats were killed, brains were removed (three brain samples put together and homogenized as one sample in each case) and dissected in the following rat brain regions: hypothalamus (H), striatum (S), brain stem (BS), midbrain (MB), frontal cortex (FC), left hemisphere (HL), and right hemisphere (HR). Three control rats were killed in parallel to both treated groups post-injection of 0.5–1.0 ml of saline solution. 2.5. Sample preparation Tissue samples were weighed and then homogenized by sonication in a solution of 0.1 M HClO4 (H, S, BS, MB, and FC in a proportion of 1:20; and LH and RH in a proportion of 1:8), the pH being adjusted to 3–4 with NaOH 0.1 M, and then boiled to 60 1C for 5 min. After that, the samples were centrifuged at 16,000g (4 1C) for 15 min. Supernatants were filtered through 0.22 mm nylon filters, concentrated in a rotovapor STUART RE300 until dryness, and then redissolved in 0.003 M HCl. Redissolved samples were centrifuged in ultrafreeMC Millipore (Waters) at 5000g during 5 min. About 300 ml of the filtrate was applied to hydrogen peroxide oxidation, as describe by Lawrence et al. (1995) for STX determination by HPLC/fluorescence detection (FLD). 2.6. Determination of STX by HPLC/FLD This method was based on the hydrogen peroxide oxidation of STX described by Lawrence et al. (1995) and modified as follows: 250 ml of 1 M NaOH and 25 ml of 3% hydrogen peroxide were added to 300 ml of the standard solution or the sample obtained after extraction procedure. The mixture was allowed to react for 2 min at room temperature. About 25 ml of 99.7% acetic acid was added to stop the reaction. Finally, 20 ml of this solution was injected into the HPLC system. 2.7. Chromatographic conditions for HPLC analysis The STX was analyzed under the conditions described previously using hydrogen peroxide oxidation as described by Lawrence et al. (1995) with slight modifications. Briefly, a 20 ml pretreated sample was injected into the HPLC system using a Rheodine 7125 injection valve. The separation of
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STX was achieved using a Phenomenex Luna C18 100P reversed-phase column (250 4.6 mm2 and 5 mm particle size). The HPLC–FLD system was an Agilent System 1100, equipped with a Quaternary HPLC pump, mobile-phase degasification system and fluorescence detector. Data were analyzed by the Agilent HP chemstation software. The column was eluted with a gradient program of solvents consisting of 0–100% (v/v) acetonitrile in 0.1 M ammonium format, adjusted to pH 6 with 0.1 M HCl, as follows: 0–5% during the first 5 min, then 5–70% during the next 4 min, then 70–100% during the next 9 min, and finally, 100–0% during the last 2 min. The flow rate was maintained at 1.5 ml/min. STX was detected with a fluorescence detector with an excitation wavelength of 330 nm and an emission wavelength of 390 nm. 2.8. Calibration studies To show linearity, a calibration curve in the range of 0.05–20 ng/ml STX was obtained. Detection and quantification limits have been evaluated on the basis of signal/noise 3:1 and 10:1, respectively. Reproducibility studies were carried out by injecting a 16 ng/ml STX standard solution into the HPLC/ FLD system, parameters such as: mean, standard deviation (SD), standard error of the mean (SEM), and variability coefficient in the area peaks were also evaluated. The HPLC/FLD method once optimized was applied for the determination of STX in the samples corresponding to different regions of the rat’s brain evaluated. 2.9. STX recovery from brain samples Recovery experiments were carried out to evaluate the efficiency of the extraction method; three control brains (total brain was suspended in 10 ml of HClO4) were spiked with 40 ng/ml of STX standard. Also brain control regions were prepared, and each region was spiked with 16 ng/ml of STX standard. After 30 min, the sample pretreatment described above was applied and these studies were carried out in triplicate. 2.10. Statistical evaluation Data of STX levels in discrete brain regions are expressed by means7SEM. Statistical evaluation of
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Ar
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Fig. 1. (a) Time retention (tR ¼ 14.5) and the area of the peak of the standard after the injection of 20 ml of STX standard (16 ng/ml). (b) A chromatogram obtained with the 10 mg/kg dose group after sample preparation under the conditions depicted in Section 2.6.
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the results obtained was performed by using ANOVA followed by Student–Newman–Keuls test. 3. Results 3.1. Calibration studies Liquid chromatographic analysis with FLD was carried out under the conditions described. Fig. 1a shows time retention (tR ¼ 14.5) and the area of the peak of the standard. Fig. 1b shows a chromatogram obtained with the 10 mg/kg dose group after sample preparation. About 20 ml of the different concentration range was injected in triplicate in the HPLC system. Good linearity (R2 ¼ 0.9916) in the 0.05–20 ng/ml STX range concentration was observed (Fig. 2). Detection limit was 11.1970.11 STX pg and quantification limit was 32.4770.14 STX pg (20 ml injected respectively). Time retention (tR) and area obtained in the reproducibility of the method, tR was 14.1870.56 min, and the area peak was 2.0170.07 FLD u.a. (n ¼ 10), each sample (16 ng/ml) was injected in triplicate. 3.2. Determination of STX in the brain of rats After sample preparation, STX concentration present in the brain of the rats was measured. Due to the low levels of STX in the different samples evaluated, a preconcentration step has been required involving evaporating the samples until dryness, redissolving in 0.003 M HCl. By
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the way, increasing the sample extract volume from 100 to 300 ml (modification of Lawrence et al. (1995)), chromatographic detection was enhanced (Fig. 3). The oxidation reagent volume was not increased. Fig. 4 shows each of the regions of the brain that have been tested. 3.3. STX recovery from brain samples Relative recovery of samples spiked with 40 ng/ml of STX standard for total brain and 16 ng/ml for each one of the regions tested (% recovery7RSD with n ¼ 3) was 59.5%71.5 for total brain; 47.6%71 for H; 47.2%71 for S; 49.8%71.8 for BS; 49.5%71.9 for MB; 49.6%71.9 for FC; 52.1%72.5 for LH; and 52.3%72.5 for RH. The mean of all the samples was 49.72%71.8. The analysis of the results led us to conclude that the recovery percentage was better in the region with higher weight. Although recoveries were approximately 50%, they were considered to be acceptable to evaluate the concentration of STX in the different regions of the brain considering the complexity of the matrix employed. 3.4. STX levels in brain regions Figs. 5a–c show the STX concentration in H, S, BS, MB, FC, LH, and RH rat brain regions after systemic administration of 5 mg/kg, at observation times of 30, 60, and 120 min at conditions described
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FLD1 A, Ex=330, Em=390 (STXRATAS\04040504.D) FLD1 A, Ex=330, Em=390 (STXRATAS\04040505.D) FLD1 A, Ex=330, Em=390 (STXRATAS\04040508.D)
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in Section 2.4. STX concentration varies from 0.7670.02 to 2.3670.19 pg/mg of H; from 0.57 0.02 to 1.1870.28 pg/mg of S; from 0.2670.01 to 1.5070.15 pg/mg of BS; from 0.2770.02 to 1.387 0.04 pg/mg of MB; 0.5870.03 to 1.5170.13 pg/mg of FC; from 0.3170.01 to 0.7670.15 pg/mg of LH, and from 0.3270.02 to 0.8070.2 pg/mg of RH, in each case the first valour corresponds to 30 min and the last one corresponds to 120 min time periods. It was observed that maximum concentration STX was found in H and minimum concentration was
observed in LH and RH. A proportional increase of STX level was also observed in each region studied across the reaction time. Fig. 5d shows the STX concentration in H, S, BS, MB, FC, LH, and RH rat brain regions at 30 min after systemic administration of 10 mg/kg. STX concentration was in H 3.570.22 pg/mg; S 3.67 0.02 pg/mg; BS 3.5370.02 pg/mg; MB 2.747 0.02 pg/mg; FC 3.4570.05 pg/mg; LH 1.7770.10 pg/mg, and RH 1.5670.07 pg/mg. Significance differences (Po0.05) to which regions were
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He
FC
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Fig. 4. Diagram of the brain identifying each of the regions of the brain that has been tested (FC ¼ Frontal cortex; He ¼ Hemispheres; S ¼ Striatum; H ¼ hypothalamus; MB ¼ Midbrain; BS ¼ Brain stem).
significantly different from one another are also shown in Figs. 5a–d. The total content of STX in the whole brain with the 5 mg/kg dose was 0.5370.016, 0.7570.11, and 1.3270.22 ng/ml at 30, 60, and 120 min, respectively. These values represent 7.12%, 10%, and 17.64% in each case. As regards the 10 mg/kg dose, the total content of STX was 3.6170.33 ng, which represents 24.06% in the whole brain. Fig. 6 shows the linearity tendency in the dose answer in the different brain regions studied over time after the systemic administration of 5 mg/kg. 4. Discussion To date, the main impediment for evaluating the toxic effects of STX was the lack of sensitivity and reproducibility for the detection of STX at pg levels. Xia and Haddad (1993) used a broad range of 3H–STX concentration (up to 64 nM) to examine saturation profiles and density distribution in adult rats. They found that STX sites do not vary greatly in affinity (most Kds ¼ 2–5 nM) in various regions of the adult rat brain; STX binding distribution was very heterogeneous in the rat with a much higher density in the cortex, hippocampus, amygdala, and cerebellum than in the brainstem and spinal cord; STX sites are mostly localized in layers mainly of neurons with low density in white matter. Recently, Andrinolo et al. (1999) developed an isolation procedure coupled with post-column derivatization HPLC method that was capable of
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detecting quantities as low as one pM of STX in body fluids and tissue samples of adults cat anaesthetized and intravenously injected with low (2.7 mg of STX/Kg) and high doses (10 mg of STX/ Kg) of toxin (Andrinolo et al., 1999). They proved that the technique developed by Oshima (1995) was quite powerful. Until now, there has been no report in the literature about a method that succeeds in quantifying STX or its analogues in different brain regions of rats, after systemic administration of the toxin. In our study, a successful evaluation of STX at ppm levels in different brain regions has been achieved using a precolumn oxidation HPLC-LD method with hydrogen peroxide as oxidizing reagent. Slight modifications on the method developed by Lawrence et al. (1995) have been included to obtain the optimal conditions for this particular matrix. Good sensitivity was obtained and a good reproducibility was observed. Nevertheless, the recovery extraction procedure about spiked brain samples tested with STX was about 50%. The research of STX levels that reach the rat brain has been complicated due to the relative complexity of the matrix employed, biological tissues (Hines et al., 1993; Andrinolo et al., 1999). As soon as rats were killed, brain samples were proceeded and analyzed; nevertheless, we detected no STX, so that we combined the brain of three animals, from 1 to 3, obtaining a good response on STX determination in the different brain regions analyzed. A substantial increase about time response can be observed in the different brain regions studied with low dose of STX, the increase was proportional to the time after STX administration. We found that STX distribution was heterogeneous, and observed STX levels in all the regions studied. After 30 min following administration of STX, the highest concentration was detected in H, S, and FC with lower STX concentration in the other regions. Conversely, at 60 and 120 min, H, and FC continue to the region with the highest concentration of toxin detected; nevertheless, in BS and MB, STX concentration was increased, but in S the increase was least less. Hemispheres always present lower concentration independently, regardless of the time interval studied. It is possible that the regions with the higher STX concentration are more accessible to the toxin since the periphery and/or they are formed in layers mostly made of neurons
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with low density in white matter (Xia and Haddad, 1993); over the time, STX is distributed to other regions.
The death of rats with high dose of STX, 10 mg/ kg, after 30 min can be produced by arrest shock place (Kao et al., 1967; Nagasawa et al., 1971;
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Fig. 5. (a) Concentration of STX in different rat brain regions after 30 min of i.p. administration (5 mg/Kg). Significance difference Po0.05 respects to 1S, BS, MB, FC, LH, RH; 2H, BS, MB,LH, RH; 3H, S, FC; 4H, S, FC; 5H, BS, MB, LH, RH; 6,7H, S, FC. The values represent the mean and SEM obtained from six determinations. (b) Concentration of STX in different rat brain regions after 60 min of i.p. administration (5 mg/Kg). Significance difference Po0.05 respects to 1S, BS, LH, RH; 2H, MB, FC, LH, RH; 3H, MB, FC; 4S, BS, FC, LH, RH; 5S, BS, LH, RH; 6,7H, S,MB, FC. The values represent the mean and SEM obtained from six determinations. (c) Concentration of STX in different rat brain regions after 120 min of i.p. administration (5 mg/Kg). Significance difference Po0.05 respects to 1S, BS, MB, FC, LH, RH; 2H; 3H, LH, RH; 4H, LH, RH; 5H, LH, RH; 6,7H, BS,MB, FC. The values represent the mean and SEM obtained from six determinations. (d) Concentration of STX in different rat brain regions after 30 min of i.p. administration (10 mg/Kg). Significance difference Po0.05 respects to 1MB, LH, RH; 2MB, LH, RH; 3MB, LH, RH; 4H, S, BS, FC, LH, RH; 5MB, LH, RH; 6,7H, S, BS,MB, FC. The values represent the mean and SEM obtained from six determinations.
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Borinson et al., 1980; Chang et al., 1992), so involves both central and peripheral cardio-respiratory system components (Andrinolo et al., 1999). After STX systemic administration, STX levels in the different brain regions studied are higher than STX levels at 120 min with 5 mg/kg dose, meaning that there is a faster distribution when STX concentration is higher (near to LD50). In this case, it is observed that almost all the regions have similar STX concentration, except the hemispheres.
The STX concentration found in total brain, with the two doses, showed that a part of STX administered i.p. can cross the blood–brain barrier and produce some alterations in the central nervous system, regardless of the effects produced in the periphery. The results obtained show that the precolumn oxidation method is suitable for such particular application providing fast and sensitive information about the distribution of STX in the different rat brain regions analyzed.
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RH Fig. 6. Results obtained in pg of STX for mg of tissue in the different brain regions studied, after i.p. injection of STX (5 mg/Kg b.w.) to male rat. After 30, 60, and 120 min. Tissues were taken from the animals, processed as detailed under Materials and Methods, and subjected to HPLC/FLD analysis for STX. The values represent the mean and SEM obtained from six determinations. (a) Po0.05 significance difference respects to 30. (b) Po0.05 significance difference respects to 60. (c) Po0.05 significance difference respects to 120.
Acknowledgments Financial support from the University of Vigo, the Regional Government of Galicia, and AbKem Iberia for supplying materials and reagents for the development of this work is acknowledged.
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