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Toxicon 50 (2007) 173–179

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Involvement of carrier-mediated transport systemin uptake of tetrodotoxin into liver tissueslices of puffer fish Takifugu rubripes

Takuya Matsumotoa, Yuji Nagashimaa,�, Hiroyuki Kusuharab, Yuichi Sugiyamab,Shoichiro Ishizakia, Kuniyoshi Shimakuraa, Kazuo Shiomia

aDepartment of Food Science and Technology, Tokyo University of Marine Science and Technology,

Konan 4-5-7, Minato, Tokyo 108-8477, JapanbDepartment of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences,

The University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113-0033, Japan

Received 9 February 2007; received in revised form 8 March 2007; accepted 8 March 2007

Available online 14 March 2007

Abstract

Although puffer fish contain tetrodotoxin (TTX) at a high concentration mainly in liver, the underlying mechanism remains to

be elucidated. In the present study, uptake of TTX into the liver tissue slices of puffer fish Takifugu rubripes was investigated by in

vitro incubation experiment. When T. rubripes liver slices were incubated with 0–2000mM TTX at 20 1C for 60min, the uptake

rates exhibited non-linearity, suggesting that the TTX uptake into T. rubripes liver is carrier-mediated. The TTX uptake was

composed of a saturable component (Vmax 47.775.9pmol/min/mg protein and Km 249747mM) and a non-saturable component

(Pdif 0.033570.0041mL/min/mg protein). The uptake of TTX was significantly decreased to 0.4 and 0.6 fold by the incubation at

5 1C and the replacement of sodium-ion by choline in the buffer, respectively, while it was not affected by the presence of 1mM

L-carnitine, p-aminohippurate, taurocholate or tetraethylammonium. The TTX uptake by black scraper Thamnaconus modestus

liver slices was much lower than that of T. rubripes and independent of the incubation temperature, unlike T. rubripes. These

results reveal the involvement of carrier-mediated transport system in the TTX uptake by puffer fish T. rubripes liver slices.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Tetrodotoxin; Puffer fish Takifugu rubripes; Carrier-mediated uptake; Liver tissue slices; Pharmacokinetics

1. Introduction

It is well known that marine puffer fish, familyTetraodontidae, contain a considerable amount oftetrodotoxin (TTX) which has a potent and specificblocking action on voltage-gated sodium channel andconsequently causes severe food poisoning including

front matter r 2007 Elsevier Ltd. All rights reserved

icon.2007.03.004

ng author. Tel/fax.: +81 3 5463 0604.

ss: yujicd@kaiyodai.ac.jp (Y. Nagashima).

fatal (Halstead, 1988; Isbister and Kieman, 2005).Extensive studies on TTX intoxication of puffer fishhave been conducted and led the hypothesis that tworoutes are implicated; symbiosis or parasitism withTTX-producing bacteria and the food chain. Theformer was reported by several research groups whofound the TTX-producing bacteria from intestine ofpuffer fish (Noguchi et al., 1987; Matsui et al., 1989;Lee et al., 2000: Yu et al., 2004). The latter wassupported by the feeding experiments using Takifugu

.

ARTICLE IN PRESST. Matsumoto et al. / Toxicon 50 (2007) 173–179174

niphobles and T. rubripes (Matsui et al., 1981, 1990;Yamamori et al., 2004; Honda et al., 2005). Theseresults indicate that TTX would be absorbed throughintestine, distributed via circulating blood, transportedto various tissues and predominantly concentrated inliver and ovary of puffer fish, although the detailedmechanism of TTX metabolism in puffer fish is stillunclear. Matsui et al. (2000) found TTX bindingprotein in the plasma of T. niphobles and suggested theprotein takes part in TTX transfer and transport in thepuffer fish. Besides, Yotsu-Yamashita et al. (2001,2002) elucidated the structure of TTX binding proteinfrom the blood plasma of Fugu (Takifugu) pardalis andcharacterized the high affinity binding of saxitoxin(STX) rather than TTX to the protein.

Our previous study supported the results of in vivo

feeding experiments, that is, liver tissue slices ofT. rubripes and T. pardalis increased TTX contentwith the incubation time while those of non-toxicfishes, striped breakperch Oplegnathus fasciatus,greenling Hexagrammos otakii and black scraperThamnaconus modestus, did not, when incubated witha TTX-containing medium (Nagashima et al., 2003).In addition, the in vitro incubation experimentshowed that liver tissue slices of T. rubripes specifi-cally accumulated TTX in preference to STXs(Matsumoto et al., 2005). Based on the in vitro

incubation experiments, the uptake of TTX into livertissue slices is unlikely ascribable to TTX bindingprotein in the plasma, since blood was substituted bythe perfusion buffer before the preparation of livertissue slices (Matsumoto et al., 2005). These resultsstrongly suggest the presence of specific mechanismwhereby puffer fish liver actively and selectivelyaccumulates TTX. However, it remains to be clarifiedhow TTX is taken up and accumulated in puffer fishliver, namely, how TTX permeates through cellmembranes comprising highly hydrophobic lipidbilayers. During the course of the studies to elucidatethe dynamic mechanism for toxification of puffer fish,we investigated the uptake of TTX by liver tissueslices of T. rubripes using pharmacokinetic analysisand report here the involvement of carrier-mediatedtransport system in TTX uptake into puffer fish liver.

2. Materials and methods

2.1. Materials

Puffer fish T. rubripes and black scraper Tham-

naconus modestus were obtained alive from Tokyocentral wholesale market and transported to our

laboratory. TTX preparation was purified fromovaries of T. pardalis by ultrafiltration using YM1membrane (Millipore, Bedford, MA, USA) and aseries of column chromatography on a Bio-Gel P-2column (Bio-Rad Laboratories, Hercules, CA,USA) and a Bio-Rex 70 column (Bio-Rad Labora-tories) as reported previously (Nagashima et al.,2003), and used in the uptake assay for TTXconcentration dependence. Crystalline TTX (WakoPure Chemicals Industries, Osaka, Japan) was usedin the uptake assays for the time course and theinhibitory effect. p-Aminohippurate (PAH) andtetraethylammonium (TEA) were purchased fromSigma (St. Louis, MO, USA) and taurocholate(TCA) and L-carnitine from Wako Pure ChemicalsIndustries. All other chemicals were of reagentgrade.

2.2. Preparation of liver tissue slices

The liver tissue slices for the in vitro uptake studywere prepared according to the previous methodwith some modifications (Matsumoto et al., 2005).Briefly, the liver was carefully removed and perfusedthrough a portal vein with ice-cold oxygenated(95% O2–5% CO2) perfusion buffer (160mM NaCl,5.4mM KCl, 4.2mM NaHCO3, 0.34mM Na2HPO4,0.44mM KH2PO4, 10mM HEPES and 5.6mMD-glucose, adjusted to pH 7.4 with NaOH solution)at a flow rate of 12mL/min for 10min. The liver wassliced (1mm in thickness) with microtome blades(A22, Feather, Tokyo, Japan) and punched (8mmin diameter) with a disposable biopsy punch(Kai Industries, Gifu, Japan).

2.3. Uptake of TTX into liver tissue slices

Each round slice was pre-incubated statically withtransport buffer (160mM NaCl, 4.8mM KCl,23.8mM NaHCO3, 0.96mM KH2PO4, 1.5mMCaCl2, 1.2mM MgSO4, 12.5mM HEPES and5.0mM D-glucose, adjusted to pH 7.4 with NaOHsolution) at 20 1C for 5min. The uptake study wasstarted by putting the slice into each well of a24-well plate (Becton Dickinson and Company,Franklin Lakes, NJ, USA) and adding 500 mLof the transport buffer containing 50 mM TTX.Incubation was carried out at 20 1C for up to90min. At time intervals, the liver slices wererapidly removed from the incubation mediaand rinsed with the ice-cold transport bufferto terminate the uptake of TTX. In another series

ARTICLE IN PRESST. Matsumoto et al. / Toxicon 50 (2007) 173–179 175

of experiment to examine the effect of TTXconcentration in the media on the uptake, theliver slices were incubated with 500 mL of thetransport buffer containing 0–2000 mM TTX at20 1C for 60min.

2.4. Inhibition assay of TTX uptake into liver tissue

slices

Inhibitory effect of substrates of typical hepatictransporters on TTX uptake into the liver tissueslices was investigated. The inhibitors were 1mML-carnitine, 1mM PAH, 1mM TCA or 1mM TEA.After being pre-incubated as described above, theliver tissue slices were incubated with 500 mL of thetransport buffer containing 50 mM TTX and eachinhibitor at 20 1C for 60min. To examine the effectof sodium-ion on TTX uptake activity, NaCl andNaHCO3 in the transport buffer were replaced bycholine chloride and choline bicarbonate, respec-tively. Besides, the incubation temperature waslowered to 5 1C to examine the temperaturedependence of TTX uptake.

2.5. Determination of TTX

TTX accumulated in liver slices was extractedwith 0.1% acetic acid by ultrasonication for 1minand heating in a boiling water bath for 10min onthe basis of the standard assay for TTX (Kodamaand Sato, 2005). The extract was defatted withdichloromethane and centrifuged at 1370g for10min. The resulting supernatant was ultrafilteredthrough a Vivaspin 500 (MWCO 5000, VivaScienceAG, Hannover, Germany) and the filtrate wasanalyzed for TTX by LC/ESI-MS. LC/ESI-MSanalysis was performed on an alliance ZsprayTM

MS 4000 LC/ESI-MS system (Waters, Milford,MA, USA). The analytical column was a CapcellPak C18 AQ (1.5� 250mm, 3 mm particle size,Shiseido, Tokyo, Japan) and maintained at 25 1C.The mobile phase consisted of 15mM heptafluor-obutylic acid in 10mM ammonium acetate (pH 5.0)and was eluted at a flow rate of 0.10mL/min.The eluate was induced into the ion source ofESI-MS and ionized by the positive ion modewith desolvation temperature at 350 1C, ionsource block temperature at 100 1C and conevoltage at 45 kV. TTX detection at m/z 320corresponding to the protonated molecular ion(M+H)+ was achieved using the selected ionrecording mode.

2.6. Determination of protein

Protein was determined by the method of Lowryet al. (1951) using bovine serum albumin as astandard.

2.7. Pharmacokinetic and statistical analyses

Kinetic parameters for TTX uptake were calculatedby fitting the uptake rate to the following equation(Michaelis–Menten equation) by means of the non-linear least-squares regression analysis using a MUL-TI program (Yamaoka et al., 1981) and the DampingGauss Newton Method algorithm.

v ¼V max � s

Km þ sþ Pdif � s,

where v and s are the uptake rate of TTX (pmol/min/mg protein) and the TTX concentration in the media(mM), respectively. Km, Vmax and Pdif are Michaelisconstant (mM), the maximum uptake rate of TTX intoliver slices (pmol/min/mg protein) and the non-specificuptake clearance (mL/min/mg protein), respectively.The determination of TTX was carried out at least intriplicate on individual experiment. Statistical signifi-cance of differences in TTX contents was evaluatedusing the Student-t test. All data were presented as themean7standard error (SE); significance was achievedwhen p was o0.05.

3. Results

The time profile of TTX uptake by the liver tissueslices is shown in Fig. 1. When incubated with thetransport buffer containing TTX, puffer fish livertissue slices obviously accumulated TTX as reportedpreviously (Nagashima et al., 2003; Matsumotoet al., 2005). In the experiment where TTX concen-tration in the media was 50mM, TTX content was134714pmol/mg protein even 5min after incubationand significantly increased with the incubation time to977777pmol/mg protein at 90min.

The TTX uptake rate (pmol/min/mg protein) intopuffer fish liver slices was assessed with variousconcentrations of TTX in the transport buffer at20 1C for 60min. As shown in Fig. 2A, the uptakerates were dependent on TTX concentration in themedia and exhibited a non-linear curve, suggesting theinvolvement of a saturable process in the TTX uptakeby puffer fish liver slices. Eadie–Hofstee analysisrevealed that the TTX uptake was composed of asaturable process and a non-saturable process, since

ARTICLE IN PRESST. Matsumoto et al. / Toxicon 50 (2007) 173–179176

the two-phase curve was obtained (Fig. 2B). Theapparent maximum velocity, Vmax and Michaelisconstant, Km of the saturable component wereestimated to be 47.775.9pmol/min/mg protein and249747mM, respectively. The apparent uptake clear-ance, Pdif of the non-saturable component wascalculated to be 0.033570.0041mL/min/mg protein.

Fig. 1. Time course profile of TTX uptake by the liver tissue

slices of puffer fish Takifugu rubripes. The liver slices were

incubated with the transport buffer containing 50mM TTX at

20 1C for up to 90min. Values are the mean7SE of three

different experiments performed in triplicate.

Fig. 2. Effect of TTX concentration in the transport buffer on TTX up

The liver slices were incubated with the transport buffer containing 0–

total uptake. Dashed and dotted lines indicate the saturable and th

regression analysis of the total uptake (�) in Fig. 2(A). Values are the

It is likely that the TTX uptake by puffer fish liverslices was dependent on temperature, since theuptake rate at 5 1C significantly decreased to 0.4fold of that at 20 1C (po0.05) (Table 1). Further-more, a replacement of sodium-ion by cholinemarkedly reduced the TTX uptake to 0.6 fold ofthe control (po0.05). Addition of 1mM L-carnitineto the transport buffer slightly inhibited the TTXuptake, although the difference was not staticallysignificant. None of other substrates tested such asTCA, PAH and TEA showed the inhibitory effecton TTX uptake by puffer fish liver slices.

Another incubation experiment was carried outto investigate the variation among fish species in theTTX uptake by liver tissue slices. In the experiment,the transport buffer was slightly modified; 200mMNaCl and 10mM HEPES. The uptake rate bypuffer fish liver slices at 20 1C increased in a TTX-concentration dependent manner as the same as inFig. 2A and attained to 12777 pmol/min/mgprotein at 2000 mM TTX (Fig. 3). As to blackscraper liver slices, in contrast, the uptake rate at20 1C was 2573 pmol/min/mg protein (mean7SDof one experiment) at 2000 mM TTX and almost thesame as that at 5 1C (data not shown).

4. Discussion

The present study is the first to reveal theinvolvement of carrier-mediated transport systemin TTX uptake into puffer fish T. rubripes liver. The

take into the liver tissue slices of puffer fish Takifugu rubripes. (A)

2000mM TTX at 20 1C for 60min. (B) Eadie–Hofstee plot of the

e non-saturable uptake obtained from non-linear least-squares

mean7SE of three different experiments performed in triplicate.

ARTICLE IN PRESS

Table 1

Effects of replacement of sodium with choline, temperature and

transporter inhibitors on TTX uptake into the liver slices of

puffer fish Takifugu rubripes

Compound Uptake (% of control)a

Control (20 1C) 100714

Na+ replacement 6374�

Low temperature (5 1C) 4075�

1mM TCA 100713

1mM PAH 9675

1mM TEA 10278

1mM L-carnitine 8573

aValues are the mean7SE of three different experiments

performed in triplicate.�po0.05 (Significantly different from control).

Fig. 3. Comparison of the TTX uptake into the liver tissue slices

between puffer fish Takifugu rubripes and black scraper

Thamnaconus modestus. The liver slices of puffer fish (�) and

black scraper (J) were incubated with the modified transport

buffer containing 0–2000mM TTX at 20 1C for 60min. Values are

the mean7SE of four different experiments for puffer fish and

the mean7SD of an experiment for black scraper. Each

determination was performed in triplicate.

T. Matsumoto et al. / Toxicon 50 (2007) 173–179 177

uptake of TTX into the liver tissue slices showedclear concentration dependence and comprised asaturable component (Km, 249747 mM and Vmax,47.775.9 pmol/min/mg protein) and a non-satur-able component (Pdif, 0.033570.0041 mL/min/mgprotein). The saturable process was dominant at alow level of TTX; at a Km value a saturablecomponent showed 74% of the total TTX uptake

and at 1175 mM TTX it was comparable to a non-saturable component. In nature, it is thought thatT. rubripes exclusively takes up TTX from circulat-ing blood to liver using this saturable mechanism,since TTX concentration in the blood of puffer fishwas negligible and less than 10mouse unit/mL,equivalent to 6.3 mM TTX (Hashimoto, 1979;Halstead, 1988).

Liver selectively transports endogenous andexogenous compounds from the circulation toaccumulate, metabolize or detoxify them usingvarious transport systems. Hepatic uptake oforganic anions, organic cations, organic solutesand bile salts is mediated by transporter proteinssuch as organic anion transporting polypeptides(OATP), organic anion transporter (OAT), organiccation transporter (OCT), organic solute transpor-ter (OST) and sodium taurocholate cotransportingprotein (NTCP) (Faber et al., 2003; Petzinger andGeyer, 2006; Shitara et al., 2006). However, to ourknowledge, there are no reports concerning thecharacterization of carrier-mediated uptake inpuffer fish liver. The TTX uptake by T. rubripes

liver tissue slice was temperature-dependent andsodium-ion dependent (Table 1). Whereas the TTXuptake was barely affected by TCA, PAH and TEAwhich are the typical substrates of NTCP, OAT andOCT, respectively (Inui et al., 2000; Geyer et al.,2006), it was slightly depressed by L-carnitine (Table1). It is possible that organic cation/carnitinetransporter is involved in TTX uptake, since TTXhas a pKa 8.76 and is amphoteric in a neutral pH(Goto et al., 1965). These results indicate that TTXis taken up through cell membrane of pufferfish liver by the active transport mechanism thatis dependent on sodium-ion gradient. UnlikeT. rubripes, the liver slices of black scraperT. modestus, non-toxic fish, showed a low uptakeactivity (Fig. 3) and independence of incubationtemperature, suggesting that carrier-mediated up-take of TTX is limited to puffer fish and other TTX-bearing animals. Further study is needed to identifythe responsible TTX transporter.

It should be noted that T. rubripes liver selectivelyaccumulate TTX but not STXs (Matsumoto et al.,2005), suggesting that TTX transport system of T.

rubripes liver clearly discriminates TTX and STXs.On the other hand, several species of tropicalmarine puffer fish (genus Arothron) and fresh waterpuffer fish (genus Tetraodon) in Bangladesh andThailand were demonstrated to contain a significantamount of paralytic shellfish toxins (PSTs) including

ARTICLE IN PRESST. Matsumoto et al. / Toxicon 50 (2007) 173–179178

STX as well as TTX (Kungsuwan et al., 1997;Zaman et al., 1997; Sato et al., 1997, 2000;Nakashima et al., 2004). These fish are assumed topossibly use distinct transport system(s) that med-iates simultaneously or independently the uptake ofTTX and PST. Furthermore, puffer fish possess avariety of TTX analogues (Yotsu-Yamashita, 2001;Yotsu-Yamashita et al., 2005). It is uncertainwhether a complicated toxin composition in pufferfish simply reflects that of their prey or is attributedto the biotransformation of the toxins in puffer fishbody. Investigation of the selectivity of the TTXtransporter by the means of in vitro uptake assayusing TTX analogues might be important and usefulto solve the above question and elucidate themetabolism of TTX in puffer fish.

Acknowledgments

This study was partly supported by the SKY-LARK Food Science Institute and the SasagawaScientific Research Grant from the Japan ScienceSociety.

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