First publ. in: Toxicology and Applied Pharmacology 234 (2009), 2, pp.

247–255

Oatp-assodated uptake and toxicity of microcystins in primary murine whole

brain cells
D. Feurstein. I<. Hoist, A. Fischer, D.R. Dietrich :;:
Human and fllvironrnen cal To.licology, University of Konscanz. Konscanz. Gennony

ABSTRACT

Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly
neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear
to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on
symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brain-
barrier (BBB) and blood-cerebrospinal-f1uid-barrier (BCFB ) it is hypothesized that MCs can be transported
Keyword"
across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition
Cyanobacteria of protein phosphatase (PP). To test these hypotheses. the presence of murine Oatp (rnOatp) in primary
Toxin murine whole brain cells (mWBC) was invt'stigated at the mRNA and protein level. MC transport was tested
Microcystin by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LFJ with/without co-incubation with the
Protei n phosphatase OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was
Oatp/OATP demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays.
Neurotoxicity All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener
Primary whole brain cells followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h
exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC
(50 and 500 pM) and BSP (50 pM ). MC-Western blot analys is demonstrated a concentration-dependent
accumulation of MCs. In conclusion, the in vieTO data support the assumed MC-congener-dependent uptake
in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

Introduction PP5 (Hastie et al.. 2005). As a consequence of PP inhibition numerous

cellular proteins e.g. intermediate filaments. are hyperphosphory-
Contamination of natural waters by cyanobacterial blooms repre- lated. thereby leading to the collapse of the cytoskeleton and loss of
sents a worldwide problem, causing serious water pollution and cellular integrity (Eriksson et al.. 1989 ; Wickstrom et al.. 1995; Batista
health hazards to humans and livestock. Human health problems are et al.. 2003 ). Cellular necrosis and apoptosis is observed in a dose- and
most likely as.s ociated with chron ic exposure to low microcystin (MC) time dependent manner. whereby apoprosis is observed at lower
concentrations in poorly treated drinking water. contaminated food . concentrations than overt necrosis (Mankiewicz et aI., 2001; Fladmark
e.g. fish. water snails, prawns, etc.. and with the intentional et aI., 2002; Gehringer, 2004; Fu et al.. 2005; Weng et al.. 2007). Due to
consumption of Aphanizomenon jlos-aquae (AFA)-based Blue-Green their structure and size MCs do not readily penetrate the cell
Algae Supplements (BGAS), shown to be contaminated with cyano- membrane via simple diffusion but rather require the presence of
toxins. specifically MCs, (Schaeffer et al.. 1999; Gilroy et al.. 2000; multi-specific organic anion transporting polypeptides (rodent Oatp/
Lawrence et at, 2001 ). MCs are the most commonly found group of human OATP) for active uptake ( Runnegar et al.. 1991; Fisc.her et aI.,
cyclic heptapeptide cyanotoxins with molecular weights ranging 2005; Komatsu et al.. 2007; Monks et al.. 2007). ,It is thus not
between 900 and 1100 Dalton (Da). represent more than 80 structural surprising that co-incubation of MCs. e.g. MC-lR. with the known
variants differing in the two variable L-amino acids (Meri'l uoto and Oatp/OATP substrates choline. taurocholate (TC) and bromosul -
Spoof. 2008 ). fophthalein (BSP) reduces the uptake of MC in vitro (Runnegar et al..
The in vivo and in vitro toxicity of MCs is primarily governed by the 1995; Fischer et al.. 2005; Komatsu et al.. 2007; Meier-Abt et al.. 2007;
potent inhibition of serine/threonine-specific protein phosphatases Monks et al.. 2007). Moreover. a knock-out mouse lacking expression
(PPs ) (Eriksson et al.. 1990; MacKintosh et al.. 1990; MacKintosh and of mOatp1 b2 in the liver presented with no overt liver pathology
MacKintosh. 1994; Toivola et al.. 1994). specifically PP1. PP2A. PP4 and when exposed to MC-lR (lu et al.. 2008). while the wild-type
counterpart showed the typical hepatotoxicity observed in i.p. or oral
• Corresponding author. Fax : +49 7531883170. MC- LR in vivo exposure experiments with mice (Ito et al.. 2000). Oatp/
E-mail address:daniel.dietrich@uni-konstanz.de (D.R. Dietrich). OATP are primarily expressed in enterocytes. hepatocytes and renal

Konstanzer Online-Publikations-System (KOPS)

URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-107126
URL: http://kops.ub.uni-konstanz.de/volltexte/2010/10712/
248

epithe,lial celJs (Kullak-Ublick et aI., 1994: Bergwerk et aI., 1996: Abe OATPl B3, while the mOatp lcl belongs to the OATPl C family with the
et aI., 1999: Ktinig et aI., 2000a: Ktinig et aI., 2000b: Kullak-Ublick human OATPl Cl. However, in contrast to the known transporting
et aI., 2004: Mikkaichi et aI., 2004: Sai et al" 2006: Naud et ai., capabilities of the human OATP (IA2, lBl, lB3) (Fischer et al.. 2005:
2007: Tani et ai., 2008) as well as in the heart, lung, spleen, Komatsu et al" 2007: Monks et aI., 2007), it is currently not known
pancreas, brain and the blood-brain-barrier (BBB) (Hagenbuch and whether or not the latter also applies to human OATPl Cl. The most
Meier, 2003: Hagenbuch and Meier, 2004). Consequently, the recent comparison of the skate Oatpldl, demonstrated to be able to
systemic distribution of MCs is governed by the degree of blood transport MC- LR at a low level, with other OATPS of the OATP family
perfusion and the type and expression level of Oatp/OATP present tree (Hagenbuch and Meier. 2004), suggests that the skate Oatpldl is
in a given organ. Due to the first-pass effect, high blood perfusion an evolutionarily ancient precursor of the mammalian-liver OATPl B
and high expression level of multiple Oatp/OATP types. MCs are family, however exerts the highest degree of homology (50.4% amino
often characterized as hepatotoxins. although other organs may acid sequence identity) with the human OATP1Cl of the OATP1C
also be affected. Indeed. Fischer and Dietrich (2000a. 200ob) family (Meier-Abt et aI., 2007). Based on the degree of evolutionary
treated carp (Cyprinus carpio) with a single dose of 400 ~lg/kg bw conservation of mOatp, as denoted by the high amino acid sequence
MC-LR and demonstrated pathological changes in the hepatopan- identity with human OATP demonstrated to being capable of
creas and kidney as well as the presence of MC-LR in several transporting MC, it was assumed that mouse Oatp have similar MC
organs including the brain 48 h post toxin application (Fischer and transporting capabilities. Indeed, the latter assumption is at least
Dietrich. 2000a). Immunoblotting of brain homogenates with anti- partially corroborated by Lu et al. (2008), who demonstrated lack of
MC-LR antibody revealed a band with molecular weight of acute MC-induced hepatotoxicity in mOatpl b2-knock-out mice.
approximately 38 kDa, corresponding to the catalytic subunits of Moreover. as mouse Oatplal , la4, las, lcl, 2bl. and 3al were
PPl and PP2A (37 kDa), thus corroborating the interaction of MC-LR demonstrated to be expressed (mRNA level) in the mouse brains
with PPs in the brain. A comparable study with mice demonstrated the (Hagenbuch and Meier, 2004: Cheng et aI., 2005), the question was
rapid appearance of MC-LR in the brain. 45 min and 60 min after the raised whether one or more of these mOatps, i.e. 1al. 1as, 1cl, and 3al,
initial intraperitoneal (Lp.) or peroral (p,o.) administration. respec- could be involved in transporting MC into neuronal cells. Mouse
tively (Meriluoto et al.. 1990: Nishiwaki et ai'" 1994). As well as Oatpslb2 and 6dl were included in the analysis as mOatplb2 was
detection of MC, pathological changes of the brain anatomy were assumed to be primarily expressed in the Jiver (Cheng et aI., 2005: Lu
observed in five day old progeny of mice wh ich had been treated with et aI., 2008) while no knowledge on brain expression was available for
a toxic extract of Microcystis aeruginosa. Of these neonatal mice 10% mOatp 6dl.
showed a reduced brain size and some presented with extensive In order to test the hypothesis that MCs are taken up actively into
pathology in the outer region of the hippocampus (Falconer et al.. neuronal cells in a mOatp-associated and MC-congener-dependent
1988). The above data suggest that as a consequence of the high blood manner. the presence of mOatp in primary murine whole brain ceUs
perfusion of the brain, significant amounts of MC could reach the brain (mWBC) was verified at the mRNA and protein level. MC transport and
across the BBB and induce brain pathology. Indeed. the above neuronal toxicity was tested with three MC congeners (MC-LR, -LW,
hypothesis is supported by the tragic events in February 1996, -LF) with/without co-incubation with the OATP/Oatp substrates TC
where 131 patients at a hemodialysis clinic in Caruaru. Brazil. and BSP. Uptake of MCs was demonstrated indirectly via cytotoxicity
employing poorly treated drinking water and overly used dialysis measurements and directly via MC-Western blot analysis, protein
cartridges. were exposed intravenously to varying concentrations of phosphatase inhibition determination, and via MC-specific immuno-
MC-congeners (MC-YR/-LR/-AR) Uochimsen et al.. 1998: Carmichael cytochemistry in mWBC cultures exposed to MCs.
et aI., 2001: Azevedo et aI., 2002: Soares et al" 2006). Of these MC-
exposed patients (mean approximate value of 19.5 pg MCcquiv/1 in Materials and methods
dialysis water (Carmicbael et al.. 2001)), 89% developed immediate
signs of neurotoxicity (e.g. dizziness, tinnitus, vertigo, headache, Chemicals and reagents
vomiting. nausea, mild deafness. visual disturbance and blindness)
with a later onset of overt hepatotoxicity and finally succumbed to All chemicals, unless otherwise stated were of the highest
multi-organ failure. Of the patients exposed 76 patients died within analytical grade commercially available. Individual MC-congeners
lO-weeks of initial intravenous exposure (Pouria et al.. 1998). (MC -LR, -LW and -LF) were obtained from Alexis Biochemicals. Lausen.
Different Oatp/OATP types appear to have varying affinities for Switzerland: Okadaic acid (OA) from Sigma-Aldrich, Taufkirchen,
MCs. Indeed, uptake of MC-LR in vitro via liver-specific OATPJ.Bl, 1B3, Germany.
Oatplb2 (mouse, rat), Oatpldl (skate) as well as the more widely Ham's F12 medium (FI2 ). minimal essential medium (MEM),
distributed (kidney, liver. BBB) OATP1A2 has previously been lscove's modified DMEM (IMDM). fetal bovine serum (FBS), penicillin/
described (Fischer et aI., 2005: Komatsu et al., 2007: Meier-Abt et streptomycin and G-418-sulphate (Geneticin) w~re purchased from
aI., 2007; Monks et al.. 2007; Lu et aI., 2008). Thus the observed liver PM Laboratories, Pasching. Austria and poIY-L-lysine, trypsin, trypsin
failure in the Caruaru incident was most likely a direct consequence of inhibitor and bovine serum albumin (BSA) were obtained from Sigma-
the liver-specific uptake of MCs via OATP. e.g. OATPl Bl and 1B3 Aldrich, Taufkirchen, Germany.
3L
(Fischer et aI., 2005: Komatsu et aI., 2007: Monks et aI., 2007), [ p]_ATP and Amersham ECL Plus Western blotting detection
whereas the immediate neurotoxicity may be explained by OATP- reagents were purchased from GE Healthcare, Munich. Germany:
mediated transport. e.g. OATP1A2 (Fischer et al.. 2005 ). ofMCs across Adenosine s'-triphosphate disodium salt, phosphorylase b from rabbit
the BBB. Indeed. OATP1A2 is highly expressed in endothelial cells of muscle and phosphorylase kinase from rabbit muscle from Sigma-
the BBB, epithelial cells of the bl~ood-cerebrospinal - nuid - barrier Aldrich. Taufkirchen, Germany and protease inhibitor cocktail set III
(BCFB) and in the cell membrane of human neurons (Kullak-Ublick was obtained from Calbiochem, San Diego. U.5.A.
et aJ., 1995: Gao et aI., 2000: Gao et aI., 2005: Lee et aI., 2005: Nies, For reverse rranscription-Polymerase Chain Reaction (RT-PCR), M-
2007). MuLV RT (1000 u), 5x reaction buffer (supplied with M-MuLV RT),
Of the mouse Oatps. mOatp l al, mOatpla4. mOatplas, and Oligo( dT) rH primer (100 pM), random hexamer primer (100 pM), dNTP
mOatpla6 belong to the same OATPM family, i.e. having greater Mix (10 mM), Ribonuclease Inhibitor (RiboLock, 2500 u) and for PCR
than 60% amino acid sequence identity, as the human OATP1A2 the 2x PCR Master Mix were obtained from Fermentas, St. Leon-Rot,
(Hagenbuch and Meier, 2004). Similarly the mOatpl b2 belongs to the Germany. Primer pairs were purchased from MWG-Biotech. Martins-
same OATPl B family as the rat rOatpl b2 and the human OATPl Bland ried, Germany.
 249

Specific pathogen-free Balb/c mice were obtained from The mWBC were cultured for 7- 8 days (70-80% confluency) and
Jackson laboratory. Bar Harbor. U.s.A. and held at the animal facility. incubated for 48 h with the toxins. MC- lR. -LW and -'LF were dissolved
University of I(onstanz. Germany. Euthanasia and organ removal was in 7S% methanol (MeOH). TC. BSP and OA in water.
carried out in accordance with the German Animal Protection Law. Incubation with 70 nM OA served as positive control (0%survival)
approved by the relevant German authority. the Regierungsprasidium in cytotoxicity studies. mWBC incubated with culture medium on'ly
in Freiburg. Germany (registry number: T-07 OS). served as negative control (100%survIval). The concentration of MeOH
never exceeded 0.5%. which served as solvent control in all experi-
Isolation. cell cultllre and characterization oJ primary murine WBC ments. No differences in viability. condition or growth rate could be
identified between solvent and negative control. For co-incubation
Six-to seven-day-old pups were decapitated and whole brains studies a single MC-congener was incubated with TC (ACROS. Geel.
were immediately removed. cut into small pieces and then trypsinized Belgium) or BSP (Sigma-Aldrich. Taufkirchen. Germany) in excess (SO
in HIB solution (120 mM NaOI. S mM I(CI. 2S mM HErES. 9.1 mM and SOD pM) for 48 h. TC and BSP alone demonstrated no reduction of
Glucose ) containing 2.S gfl trypsin for 12 min at 37 °C. Trypsin cell viability at the concentrations used (data not shown).
inhibitor (3.7S g/I) was added and mWBC were centrifuged for 5 min
at 300 xg. The supernatant was discarded and the resulting pellet was Detennination oJ MC uptake in mWBC via Western blot analysis (WB)
re-suspended in culture medium (1: I (v/v) IMOM/FI2. supplemented and immunocytochemistry (lCC)
with 10% heat inactivated FBS and 1% penicillin-streptomycin). Cells
were gently triturated with a I.S inch 21 gauge needle and then Western blot. The medium of MC-exposed mWBC was removed.
filtered through a 100 IJI11 nylon mesh. The dissociated rnWBC were cells were washed 3 times with phosphate-buffered saline (PBS) and
seeded in poIY-L-lysine (SO mg/l) coated 6 and 96 well plates (Greiner homogenized in 60 pi ice-cold extraction buffer (W mM
Bio-One. Frickenhausen. Germany) and 8 well chamber slides (Bio- triethanolamine (Tris)-base. pH 7.S. 140 mM NaCI. 5 mM EDTA.
Coat; BO. Heidelberg. Germany ) at a density of 1.6x lOCi cells/ml and 0.1%(v/v) Triton X- lOO. Ix protease inhibitor cocktail). To remove cell
cultured at 37 °C and 5%CO2 . Culture media was renewed every 72 h. membrane fragments. the lysate was centrifuged for S min at
To characterize the primary murine whole brain cells (mWBC). 16.000 xg and 4 0c. The protein content was determined by the
Western blots were carried out using brain cell-type specific method of Bradford (Bradford. 1976) (Bio-Rad Protein Assay; Bio-Rad.
antibodies kindly provided by Prof. leist (Ooerenkamp-Zbinden Munich. Germany) and equal protein amounts (30 ~Ig/Iane) were
Chair of Alternative in-vitro Methods. University of Konstanz ). Briefly. separated using a 10% sodium dodecyl sulfate-polyacrylamide gel
following 8 days of in vitro cultivation. the presence of neurons. electrophoresis (SOS-PAGE) according to the method of Laemmli
astrocytes. microglia. endothelial cells and fibroblasts in the mWBC (1970) at constant 200 V. Following electrophoresis. proteins were
culture. as well as in homogenates of whole brains of six-day-old pups. transferred onto nitrocellulose membranes (Whatman. Oassel.
was assessed via Western blot using antibodies to anti-class III 1"'>- Germany) at 300 mA for 90 min as previously described (Tobwin
tubulin (1 :1000; Covance. Emeryville. U.s.A; Fig. lA ). anti-glial et al.. 1979). Membranes were incubated with blocking buffer (HBS
fibrillary acidic protein (1: 1000; Sigma-Aldrich. Taufkirchen. Ger- - Tris-buffered saline with Tween 20 (100 mM Tris-HCI. 0.9% (w/v)
many; Fig. IB ). anti-F4/80 (I : 1000; AbO serotec. Martinsried. NaCl. pH 7.6. 0.1% (w/v) Tween 20) containing 1% bovine serum
Germany; Fig. 1C) and anti-vimentin (1: 1000; developmental studies albumine (BSA) for 30 min at RT and incubated with polyclonal
hybridoma bank. university of Iowa. U.5.A). Positive signals were rabbit anti-MClR#2 (1 :SOO) (Mikhailov et al.. 2001). monoclonal
observed in mWBC for neurons. astrocytes and microglia (Figs. lA- C). mouse anti-AOOA ( I :700) (cIone A04G2. Alexis Biochemicals.
vimentin. representing endothelial cells and fibroblast type cells. lausen. Switzerland). monoclonal mouse anti- PP1 et (1:500)
could not be detected (figure not shown). corroborating earlier (Sigma-Aldrich. Taufkirchen. Germany ). monoclonal mouse anti-
fiindings in mWBC (Bologa et al.. 1983). PP2A/C (1: 1000) (Upstate. Temecula. U.5A) and monoclonal mouse
anti-GAPOH (1 :30.000) (Sigma-Aldrich. Taufkirchen. Germany) for
16 h at 4 0c. Secondary antibodies were horseradish peroxidase
(HRP)-conjugated mouse anti-rabbit- ( I :SO.OOO) (Sigma-Aldrich.
A 2
Taufkirchen. Germany) and goat anti-mouse (1 :20.000) (Sigma-
anti-class lilt p-Tubulin ~I'tr~~~~.~~~s~1 50kDa Aldrich. Taufkirchen. Germany) antibodies. Immunopositive bands
were visualized via ECl substrate according to the manufacturer's
anti-GAPDH 1 I.- 37kDa recommendations and the resulting chemiluminescent signal was
detected using Fujifilm LAS-1000 (FUJIFllM Electronic Imaging.
2 Kleve. Germany). For re-probing nitrocellulose membranes. blots
B
anti-GFAP I, " 1- 55kDa
were stripped using SO ml of SO °C stripping buffer (SO mM Tris-
base. 2% (w/v) SOS. pH 6.8. 100 mM (',-mercaptoethanol) for 30 min
anti-GAPDH 1
..._____-__.......1- 37kDa under gentle shaking.

c Immunocytochemistry. The medium of MC-exposed mWBC was

1
anti-F4/BO 1- 160kDa removed. cells were washed 3 times with PBS and fixed in - 20 'c cold
~:::::::::::==~ ethanol/acetone (1: 11) for 2 min on ice. The fixation buffer was
antl-GAPDH I 1- 37kDa discarded and the chamber s'lides were air dried for 30 min and stored
at - 20 °C. For the detection of MC. mWBC were re-hydrated in PBS and
fig. 1. Characterization o/" mWBC using cell type-speci~c antibodies and Western blot. incubated in blocking buffer containing 1% BSA for 30 min. Rabbit
(A) Neuron-s pecific class IIl lHubulin antibody in mWBC(lane 1. 15 , Ig total protein ) and anti-MClR#2 antibody (I :SOO) (Mikhailov et al.. 2001) was applied in
murine brain homogenate (lane 2. 15 ,'g tot,,1 protein). (8) Astrocyte-specific glial a humidified atmosphere and incubated for 60 min at RI. The
~b rill.lty acidic protein (CFAP) antibody in mW8C (lane I. 20 pg tot,,1 protein) and
murine brain homogenate (lane 2. 20 flg total protein ). (e) MCA497C ,lntibody against
fluorochrome-conjugated secondary antibody (1 :SOO) goat anti-
murine microglial surface protein f 4 (80 in mWBC (lane I. 40 pg total protein) and rabbit-Alexa488 ('\"'.1' : 49S nm; Invitrogen. Karlsruhe. Germany).
murine brain homogenate (lane 2. 20 .tlg total protein ). was added and samples were incubated for 60 min at RI. For
250

cytoskeleton detection a concentration of 700 nM TRITC-phalloidin OATPI B3 (Komatsu et al.. 2007) (cultured in MEM supplemented with
(Am,x: 547 nm: Sigma-Aldrich, Taufkirchen, Germany) was used to 10% FBS. 1% penicillin/streptomycin and 400 pg/ml G418-sulphate at
stain actin filaments and nuclei we.re counterstained with 2.5 pM 37 · C and 5% CO 2 ) were employed as an internal OATP positive control.
Hoechst 33342 ('\m,x: 352 nm; Invitrogen, Karlsruhe, Germany). Culture medium was removed every 72 h until cells were used for the
Finally, culture slides were mounted with Fluorescent Mounting experiment (80-90% confluence).
Medium (DAKO, Hamburg. Germany) and visualized using a con-focal
microscope (LSM 510 META. Zeiss. Gottingen, Germany). Reverse transcription-polymerase chain reaction (RT-PCR). Each
reaction was carried out in a final volume of 21 ~t!. Therefore 6.8 pi
Detennination of MC-congener specific cytotoxic effects in mWBC of total RNA (1.3 pg). 3.2 ~t! RNase-free water. 0.5 ~t! random hexamer
primer and 0.5 ~~ oligo(dT) were incubated for 5 min at 70 · C following
Cytotoxicity assay. mWBC cell viability was assessed by deter- 5 s at 25 · C and chilled for at least I min on ice. Reaction buffer (4 ~~).
mination of 3-(4.5 dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium 2 ~t! dNTP mix. 0.5 ~t! ribonuclease inhibitor and 1.5 ~t! RNase-free water
bromide (MTT) reduction according to Mosmann (1983). Briefly, after were added (5 min. 25 · C). Finally. 2 ~t! of Reverse Transcriptase (RT) or
48 h toxin exposure. 25 ~~ MTT solution (5 mg/ml, ACROS, Geel, RNase-free water (for RT negative controls ) was incubated for 10 min
Belgium) was added to each well and incubated for 90 min at 37 "C. at 25 dc, 60 min at 37 DC following 10 min at 70 .c.
After incubation the medium was gently removed and 100 ~ PCR for mOatp/OATP was performed in 25 ~t! reaction mixtures
solubilization buffer (95%(v/v) isopropanol, 5%(v/v) formic acid) was according to Mulhardt (2002) with minor modifications. Primers were
added to each well. shaken carefully for at least IS min to re-dissolve designed (Primer3 software; Rozen and' Skaletzky. 2000) re-hydrated
the formazan product. Absorption was measured at 550 nm using a with RNase-free water to the appropriate volumes according to MWB-
microtiter plate reader (Tecan. Crailsheim, Germany). The test was Biotech data sheets (100 pmolh~). gently mixed and shaken over night
carried out independently three times in duplicates for each congener. at 4 "C. The sequences of primer pairs used in this assay are shown in
Table 1. Briefly. amplification was subsequently carried out by mixing
Radioactive protein phosphatase inhibition assay (rPPIA). Phospho- 2 pi of cDNA product with 12.5 ~~ of 2x PCR Master Mix solution, 4 ~~ of
rylation of phosphorylase b with [J2P1-ATP and the protein phos- the primer pair mix (0.5 pM forward- and 0.5 pM reverse primer) and
phatase inhibition assay were performed as described by Fischer and 6.5 ~~ of RNase-free water. Human OATPI B3 was used as a positive
Dietrich (2000c). control. PCR reaction was performed in a thermocycJer (Primus 96
In a preliminary step. cell extracts of untreated mWBC were taken Plus; MWG-Biotech. Martinsried. Germany) under the following
through a dilution series with extraction buffer (see immunoblot- conditions: initial denaturing for 3 min at 94 'c. 30 cycles of
ting) in order to determine the linear range of dephosphorylation of denaturing for I min at 94 'c. annealing for I min at 58 DC and
the [l2P1-ATP-labeled substrate. Protein concentration was deter- e.longation for I min at 72 · C followed by an additional extension for
mined according to the method of Bradford (1976). In the final assay 7 1I1in at 72 0C,
30 pg of total I?rotein in a volume of 20 pI per sample was employed.
mWBC extracts were added to 20 pi of protein phosphatase assay Agarose gel electrophoresis of PCR products. Each PCR product (20 ~t!)
buffer (consisting of 0.1 mM EDTA, 20 mM imidazole-HCI, pH 7.63. was separated by gel electrophoresis on a 3% agarose gel buffered in
I mg/ml BSA. 0.1%(v/v) I',-mercaptoethanol final concentration). The TBE (10.8 g/I Tris-base. 5.5 g/l boric acid. 4 rnl (v/v) 0.5 M EDiA ph 8.0).
reaction was started by adding 20 ~t! [32P]-phosphorylase a in DNA ladder (Gene Ruler 100 bp; Fermentas. St. Leon-Rot. Germany)
solubilization buffer (50 mM Tris-HCI. pH 7.0.0.1 mM EDTA. IS mM was employed to determine the PCR product size. Gels were stained
caffeine. 0.1 %(v/v) I') -mercaptoethanol). After incubation for 5 min at by ethidium bromide. visualized under UV light (302 nm).
30 ·C the reaction was stopped by addition of 180 ml ice-cold 20% photographed (Polaroid camera and Polaroid 667 films) and then
(w/v) trichloroacetic acid and cooling on ice for at least 10 min. scanned for documentation.
Subsequently the samples were centrifuged for 5 min at 12.000 xg. In
order to extract free [32 P1 180 ~ of the resulting supernatants were Purification and sequence confinnation of PCR products. Following
mixed with 200 .Lt! of acid phosphate [buffer (1.25 mM KH 2P0 4 in 3%agarose gel separation of PCR products. bands were excised and the
0.5 M H2 S0 4 ). 500 ~ isobutanol/heptane (1:1) and 100 ~~ ammo- PCR products purified via microcolumn elution (QIAquick gel
niumbeptamolybdate (5% (w/v)) by vigorously vortexing. Radio- extra.ction kit: Qiagen. Hilden. Germany). Purified PCR products
activity was counted in a liquid scintillation counter (tS 6500; were sent to GATC (Konstanz. Germany) for DNA sequencing.
Beckman Coulter. Krefeld. Germany) after mixing 300 ~~ of the [3 2 P1_
containing solvent layer with 1 ml of scintillation cocktail (Ready
Safe: Beckman Coulter. Krefeld. Ge.rmany). Total ser/thr-specific Table 1
protein phosphatase activity of MC- and OA- (positive control) Primer sequences used for PCR
exposed mWBC was calculated by determining the percentage loss of
Gene Primer Sequence 5· .... 3' Product size Degree of
radioactivity from untreated cells (negative control = 100% activity). (protein) (base pairsl alignment
Each sample was analyzed three times in duplicate. The degree of (BlAST) (%)
protein phosphatase inhibition expressed as activity of the corre- Slco/c/ {(Mplel) Sense gtagggganeeagetecte 204 100
sponding control was normalized to the corresponding number of Antisense gcataatgageeeaaaagga
viable cells. This was achieved via division of the mean protein Slcola/ (Oatplal) Sense atceagtgtgtggggacaat 235 100
Antisense atggctgcgagtgagaagat
phosphatase inhibition with the corresponding median of cytotoxi- Slcola5 (OatplaS) Sense gcacagagaaaaagceaagg 166 100
city (MTT assay) observed. Antisense ctccaggtamgggcaaga
Slco3al (Oatp3al) Sense gccmtggtgaagaagetg 275 100
Detennination of mOatp transporters in mWBC Antisense ga.lgcaggctgacaggt agg
SIcolb2 (Oatplb2) Sense ncaccaeaacaatggccta 194 100
Antisense ttttccccaeagaeaggttc
RNA extraction. Total RNA was isolated from mWBC using the Slc06d I (Oatp6d I) Sense gaagcaggctcaggtggtag 249 n.d.
RNeasy Mini Kit (Qiagen. Hilden. Germany) according to the Antisense acgaccgctaaaaacgacag
manufacturer's instructions. Column-bound total RNA was eluted SLCOl83 (OATPIB3) Sense gggtgaatgcccaagagata 168 100
with 60 pi RNase-free water and purity was determined by the quotient Antisense attgactggaaacccattgc
260 nm/280 nm of optical density (OD). Stably transfected HEK293- n.d. : not detectable due to limited band visibility and thus PCR product extraction.
 251

A
anti-MC-LR
_
MC-LR()1M]
39koa
.-26kOa
1
-...
~100
c
0
u 75
-+-LR
-e-.LW
•• .. - · LF

anti·GAPDH ::========:
~____~__________~~~1-3~oa
~
0
•
.... 50
.,e.
B
anti·PPl
c 0.2 0.4 0.6 0.8 c/+ MC-LR

1-37.5koa
[~M]
-
>.
.s;
:e
I'll
25
••
:.: .......
.. ....
' '
.. **

anti·PP2A1C
:===========:I-36koa a..
a.. 0
**

~1--3~Da
i i
anti·GAPOH L-______________________ 0.2 0.4 0.6 0.8 1.0
[pM]
c 2 3

_~1--3~oa
Fig. 4. Total protein phosphatase activity in mWBC after treatment with MC-congeners-
anti·MC·LR LR. -LW, or -LF normalized to median of cytotoxicity. values represent mean±SD of
L-_ _ _ _ _...
three independent experiments and are expressed as percentage of control. Statistics :
one-way ANOVA with Dunnetrs post-test (P < O.OS ..... : P<O.01 ...... ).

Fig. 2. (A) Determination of intracellular MC-Lt{ after 48 h exposure ofmWBC to,] range
of MC-LR concentrations. Western blots using MC-antibody MCLR#2 (Mikhailov et al"
2001). c: control mWBC, cI+: 1 flM MC-LR added to mWBC cell lysate: (B) Determination multiple comparison test, where appropriate, using GraphPad Prism
of intracellular PPl a and PP2AIC expression in mWBC exposed to MC-LR. Western blots
4.03, P<0.05 (*). P<O.01 (**) and P<O.OOl ( ~ U) .
using PPl a and PP2A/C antibodies. (C) Determination ofPP-associ.ted MC-LR after 48 h
exposure ofmWBC. Lane 1: mWBC following exposure to 1 pM MC-LR. lane 2: purihed
rabbit muscle PPl (New England Biolabs. Germany) incubated with 1 ItM MC -LR in Results
vicro. lane 3: purified human etythrocyte PP2A (I'romega. Germany) incuba ted with
1 ftM MC-LR. Uptake of MC-LR into mWBC

Sequence results received were aligned (htrp ://blast.ncbLnlm.nih.gov/ A concentration-dependent uptake of MC-LR was observed in
bI2seq /wblast2.cgi ) with the corresponding known mOATP/OATP1 B3 mWBC exposed to MC-LR for 48 h (Fig. 2A). MC-LR-positive bands
sequences. ranged between - 39 and - 28 kOa. The - 39 kOa band corresponded
to the catalytic subunits of PP1 (37.5 kOa) and PP2A (36 ·kOa ). as
Investigation of mOarp 1b2 protein expression in mWBC via Western confirmed vi'a immunoblotting using antibodies against PP10 and
blot analysis PP2A/C (Fig. 2B). yet in addition containing a covalently bound 1 kOa
MC. The latter was also confirmed by MC-incubation studies using
For analysis of mOatp l b2 via WB, mWBC were cultured and purified PP1 and PP2A, which provided a comparable - 39 kOa band
homogenized as described above. Briefly, the mWBC homogenate was (Fig.2C).
centrifuged for 40 min at 16,000 xg and 4 · C in order to obtain the
crude membrane fraction . The pellet was resuspended and protein Intracellular localization and cytotoxic effects in mWBC
concentration was determined by the method of Bradford (1976) (Bio-
Rad Protein Assay ; Bio-Rad, Munich, Germany ). Proteins were Incubation (48 h) of mWBC with 0, 0.6!JM and 5.0 ~tM MC-L.R and
separated by SOS-PAGE, blotted on a nitrocellulose membrane and subsequent immunofluorescent analyses using con-foca~ microscopy
incubated with an antibody against mOatp 1b2 (Lu et aI., 2008), kindly demonstrated the presence of MC-LR in the cytosol and the nuclei of
provided by Prof. Klaassen (Department of Pharmacology, Toxicology mWBC (Figs. 3B and C). Loss of structural integrity of the cytoskeleton
and Therapeutics, University of Kansas Medical Center, Kansas, USA). was MC-LR concentration-dependent. as shown by the congregation
of actin filaments around t he nuclei (Figs. 3B and C). Loss of
Statistics cytoskeleton integrity also coincided with protein phosphatase
inhibition (Fig. 4) but not directly to cytotoxicity as determined via
Statistical analysis was performed by one-way analysis of variance MTT assay (Fig. 4). However, complete congregation of actin filaments
(ANOVA) with Ounnett's post test. Bonferroni or Newman-Keuls around the nuclei (Fig. 3C) as observed following incubation with 5 !JM

Fig. 3. Intracellular MC-LR locali zat ion and associated cytoskeleton abnormalities in mWBC exposed to MC- LR for 48 h. Immullolabeling of MC-LR (green : MCLR#2-Alexa488 ). actin
filJments (red: TRiTe-Phalloidin) and nucleus (blue: Hoechst 33342); (A) control mWBC; ( B) mWBC exposed to 0.6 flM MC-LR ; (C) mWBC exposed to 5 flM MC-Lit Scale b;}r to flITl.
252

120 3

i 100
··.·· MC·lF
!0 ........! ........! ... anli·Oalp Ib2
u
80 •• • "''1" "
'0 ".I:.;r
60 .. r·········:~~i ..
~ ..
'\r...
,
~ 40 Fig. 7. Western blot ofmOatplb2 in mWBC crude membrane fractions using an anti·
i Oa tplb2 polyclonal antibody (lu et al.. 2008). lane I : mWBC (40 fill): lane 2: murine

">" 20 brain homogenate (40 pg): lane 3: murine liver homogenate (positive control. 40 ~g).

0 ~ and BSP Uacquemin et al.. 1994: Runnegar et al.. 1995; Kanai et al..
I i i i i "I i i i , i'l 1996: Fischer et al.. 2005 : Monks et al.. 2007 ). The viability of mWBC
0.1 1 10
exposed to MCs was compared to mWBC exposed to the combination
concentration (IIM)
or 50 and 500 pM TC (Figs. SB. C. E. F. H, I) or 50 pM BSP (Figs. SA. D, G)
Fig. S. Cytotoxicity in mWBC following 48 h exposure to MC· lR. ·lW, and ·IF (conc. and the corresponding MCs. Co·incubation ofTC and BSP with MCs
range 0.1- 5 ~tM ), determined via MTf. Viability values represent mean±SEM of three generally resulted in a reduction of observed cytotoxicity. For MC· LR
independent experiments and are expressed as percentage of control. Statistics: this reduced cytotoxicity was observed at ~ 5 pM MC· LR only, whereas
one·way ANOVA with Dunnett's post·test (P<0.05 ,..'"; P<O.Ol ....').
reduced cytotoxicity was already observed at 1 and 3 pM for MC · LW
and - LF. respectively. Moreover, while the reduction of cytotoxicity in
MC- LR corresponded to approximately 40% reduction in cell viability. MC-LW and · LF exposed mWBC co-incubated with 50 fun BSP was
as determined via MTI. limited , the corresponding experiments with 50 and 500 J.!M TC
demonstrated a much greater reduction in MC· mediated cytotoxicity.
MC-congener specific cycotoxic effect Provided that MC is primarily transported via mOatps, the data as
presented here suggest better MC transport· inhibition by TC than by
Exposure of mWBC for 4S h to MC· LF. -LW and - LR resulted in BSP, despite that the degree of reduction in MC·mediated cytotoxicity
significant inhibition of protein-phosphatase in mWBC (Fig. 4) at was comparable in the 50 and 500 J.!M TC co· incubation experiments.
~ 200 nM, ~400 nM and ~600 nM . respectively. Protein·phosphatase
TC- induced reduction of MC transport was also corroborated by
inhibition co.rresponded with the observed cytotoxicity at the highest Western blots demonstrating smaller protein bands positive for MC-
concentrations (Fig. 5). Indeed. while exposure of mWBC to 5 J.!M MC· LW in cells co-incubated with TC than in cells exposed to MC·LW only
LF resulted in a complete loss of viable cells. the same concentrations (Fig. 9 ). Moreover. the width of the MC-LW positive protein bands
of MC· LW and - LR resulted only in a 33% and 54% reduction of cell decreased in a MC- LW concentration (1.0-0.05 J.!M MC-LW) depen·
viability. respectively. dent manner, being smallest in cells exposed to 50 nM MC-LW and
500 J.!M Te.
Detennination of mOatp-associated transport of MC
Discussion
To further evaluate the role of bile acid transporters in the active
uptake of different MC congeners into mWBC. the presence of various Contamination of human water sources with the cyanobacterial
mOatps was examined at the mRNA level. mWBC were screened for toxin MC has been shown to induce hepato- and neurotoxic effects in
six mOatps (Oatpl cl . 1as . 3al. I a I. I b2 and 6d 1) and resulted in single patients of a dialysis clinic in Caruaru. Brazil (Pouria et aI., 1998). The
positive bands for five tested mOatps (Oatplcl. la5. 3al.lal and Ib2 ) early onset of neurotoxicity observed could be explained by the
with 166-275 bp corresponding to the expected size of amplified uptake of MCs via OATPS e.g. OATPIA2 which is highly expressed in
products with the primers employed (Fig. 6). Water and RT negative endothelial cells of the BBB, epithelial cells of the BCFB and in the
membrane of human neurons (KuUak-Ublick et al.. 1995 : Gao et al..
controls did not show any contamination/bands (data not shown). The
amplified products were sequenced to confirm the detection of the 2000 : Fischeret al.. 2005: Gao et al .. 2005: lee et al.. 2005: Nil'S. 2007 ).
visualized mOatp fragments. While five of the six mOatp RT-PCR However. neurotoxicity can only evolve if MCs are actively and
products could be confirmed via sequencing and alignment analysis effectively transported. e.g. via MC· transport competent Oatp/OATP.
(Table I ). there was insufficient DNA extractable for mOatp6d I or possibly other yet unknown transporters. expressed in brain cells.
(S1c06d I ) to allow sequence con fi rmation. In addition. mOatpl b2 Transport of MC- LR has been demonstrated for human OATPIA2. I BI.
was detectable in mWBC. whole brain and liver homogenates (positive I B3. rat/mouseOatpl b2 and scateOatp I d I (Fisc her et al.. 2005:
control. (Lu et al.. 200S)) at the protein level (Fig. 7) with a molecular Komatsu et al.. 2007: Meier-Abt et al.. 2007: Monks et al.. 2007: Lu
weight of70- S0 kDa (www.uniprot.org: UniProtKB/Swiss- ProtQ9JJL3 - et aI., 200S) but not for the human OATP2BI and rat Oatp lal and la4
1(501 B2_Mouse). mOatpl b2 known molecular weight 76.729 kDa). (Fischer et al.. 2005). thus demonstrating that not all Oatp/OATP are
In order to corroborate the association of mOatp with the active capable of transporting MC·LR. The latter however also raised the
uptake of MC congeners in mWBC. co-incubation studies were carried question whether different MC congeners are transported by different
out employing excess concentrations of the OATP/Oatp substrates TC Oatp/OATP albeit with varying efficiency. thereby suggesting that an
Oatp/OATP not transporting MC- LR does not exclude its capability of
transporting other MC congeners.
The results presented in this study. c.learly demonstrate the
400 expression of at least five mOatps in primary murine whole brain
cells (Fig. 6 ) at the mRNA level. The different band intensities as
300 observed in Fig. 6 most likely result from varying binding efficiencies
200 of the respective primers generated to the respective mWBC mOatp
mRNAs.
Fig. 6. RT· PCK ofOatp· mRNA expression in mWBC.lane I: Oatplal; lane 2: Oatplcl :
The detection of the liver specific MC·LR transporter Oatpl b2
lane 3: Oatpla5; lane 4: Oatp3al; lane 5: Oatp6d 1: lane 6: Oatplb2 : lane 7: OATP183 (mouse. rat ) (Fischer et al .. 2005: Lu et al.. 200S) in liver and brain
(positi ve control : stably transfected HEK293-0ATI'IB3 ). M: Gene ruler (bp). homogenates as well as in mWBC at the protein level (Fig. 7). suggests
 253

MC-LR

A
c=:::::IMC-LA
-----------------B -==::11 MC·LR
C
c:::::::::J MC-lR • SO",M le
c:::::::::::J MC-t.A • 50tlM 8SP
1:=::1 MC-LA
100 b _ 120
B, C
•. c
= MC·LA • soc.'" Te
~

ei" BO
~
b b
C-
0 ~
u
0 60
~ I. II
~
~ 40 .
.~ -
20
C 3 3 5 5 7 7 5
[,uM]

MC-LW
0 E F

100 " C = M C·LW

c:::::::::J MC-LW • so"aM 8SP
= M C· LW
c::::::::::I ,\t<> lW . SOtaMTC =
.::::::::!!I MC·LW
MC·LW . soc.'" TO
e BO e
§ §
'0 60 '0
~ ~
.~ .~
:s 40
.,~
ca
'"
'S

20
C 3 3 5 5 7 7 c 3 3 5 5
r~M] I~MI

MC-LF
G H
IIII:=MC·LF a::::II MC -LF
c:::::::::::JMC -LF. ~ MTC = "'C'LF • so.'" Te
ei" g
0
u
80
'0 '0
t.. ~
.~ .~
~,. ~,.

lJ.tM] II'M!
Fig. 8. Cytotoxic effects of MC-LR (A, B. Cl . ·LW (D, E, F), -LF (G, H. I) in mWBC co-incubated with/withou t th e Oatp substrates taurocholate (TC: 50 and 500 ~ ) and
bromosulfophthalein (BSI': 50 ~ IM ) . Contro l mWBC (C: black columns) represent 100% ceH viability, MC-<ongener treated mWBC (grey columns) and MC-co ngenerHC/BSP co-
incubated mWBC (white columns) followin g 48 h exposure. Values represent mean ±SEM of three independent experimen ts. Statist ics: one-way ANOVA with New man- Keuls
mul tip le comparison test (comparison of a: MC vs. MC. BSI'(rC; b: MC vs. control : c MC +HSP{TC vs. c.ontrol. all at P<O.05).

the functional expression of mOatpl b2 in mouse brains and mWBC. MC congeners (Fig. 8) in conjunction with the fi ndings by Lu et al.
As Oatpl b2 (mouse, rat) was demonstrated to actively transport MC- (2008) suggests that although mOatpl b2 efficien tly transports MC-LR,
LR the observed MC- mediated cytotoxi ci ty and pp inhibition in mWBC mOatpl b2 mediated MC transport may not be as important in mouse
may be at least partially due to mOatpl b2 mediated MC transport into brains and that other mOa tp, expressed at higher levels than
mWBC. However, other mOatps may also be involved in the transport mOatpl b2. as well as MC congeners other than MC-LR may be more
ofMCs, since Oatpl b2 knock-out mice have shown to develop no overt important for MC- mediated neurotoxicity. However. only specific
hepatotoxicity, albeit detectable MC-LR was observed in liver homo- expression of individual mOatp and MC- transport analyses. as
genates (Lu et al.. 2008 ). Incuba tion of mWBC with the three d ifferent discussed below. will allow determination of the contribution of
MC congeners MC- LR, - LW and -LF resulted in congener-dependent each individual mOatp expressed in mWBC and mouse brains in the
toxicity as demonstrated by the observed concentration-dependent observed MC-congener specific transport.
cytotoxicity (Fig. 5 ) and protein phospha tase inhibition (Fig, 4 ). Intracellular locali zation of MCs was proven via immunocyto-
Moreover, the observed variant cytotoxicity with the three different chemistry (Fig. 3) and Western blotting of mWBC cell homogenates
254

u Bergwerk. AJ.. Shi. X.. Ford. A.C.. Kanai. N.. Jacquemin. E.. Burk. R.D .. Bai. S.. Novikoff. P.M..
I-
:::E ~ Stiege.r. B.. Meier. P.J.. Schuster. V.L.. Wolkoff. A.w.. 1996. Immunologic distribution of
:::E
s;'"
c:> :.. an organic anion transport protein in rat liver and kidney. Am. J. Physiol. 271.
c:>
c:> G231 - GZ38.
+ '"+ Bologa. l.. Joubert. R.. Bisconte. J.c.. Margules. S.. Deugnier. MA. Derbin. C..
~ ~ le
..l ~
Herschkowitz. N.. 1983. Development of immunologically identified brain cells in
U U U U
culture : quantitative aspects. Exp. Brain Res. 53. 163-167.
:::E :::E :lE :::E Bradford. M.M .. 1976. A rapid and sensitive method fo r the quantification of microgram
:::E :::E :::E :lE quantities of protein utilizing the principle of protein-dye binding. Analytical
c
c:>
c
c:> c
c:> c:> c:> .§ Biochemi stry 72. 248-254.
d+ '"
C ~ ~
'" Carmichael. WW.. Azevedo. S.M .. An. J.S .. Molica. R.J .. Joc.himsen. E.M .. lau. S.. Rineharr.
__ 1_39kDa K.l.. Shaw. G.R .. Eaglesham. G.K .. 2001. Human fatalities from cyanobacteria:
anIi·ADDA I -26kDa
chemical and biological evidence for cyanotoxins. Environ. Health Perspect. 109.
663- 668.
Cheng. X.. Maher. J.. Chen. C.. Klaassen. C.D .. 2005. Tissue distribution and ontogeny of
1-37kDa mouse organic anion transporting polypeptides (Oatps). Drug Metab. Dispos. 33.
1062-1073.
Fig. 9. Oatp-dependent uptake of MC-lW in mWBC following 48 h co-incubation with/ 'Eriksson. J.E.. Paatero. G.l.l.. MeriJuoto. J.A.O .. Codd. GA. Kass. G.E.N .. Nicotera. P..
OITenius. S.. 1989. Rapid microfilament reorganization induced in isolated rat
without the Oatp substrate taurochol¥e (Te: 500 ~). Western blot using anti-ADDA
hepatocytes by microcystin-lR. a cyclic peptide toxin. Exp. Cell Res. 185. 86- 100.
antibody (clone AD4G2 ) for MC-lW adduct detection.
Eriksson. J.E .. Toivola. D.. Meriluoto. J.A .. Karaki. H.. Han. Y.G .. Hartshorne. D.. 1990.
Hepatocyte deformation induced by cyanobacterial toxins rellects inhibition of
protein phosphatases. Biochem. Biophys. Res. Commun. 173. 1347-1353.
(Figs. 2 and 9). thus conclusively demonstrating transport of MCs into Falconer. !.R.. Smith. J.V .. Jackson. A.R.. Jones. A.. Runnegar. M.T.. 1988. Oral toxiCIty of a
mWBC. mOatp-associated MC transport. is also supported by the bloom of the Cyanobacterium microcysCis Aenlginosa administered to mice over
fao! that uptake of MCs into mWBc. as indicated by the observed periods up to 1 year. J. Toxicol. Environ. Health 24. 291 - 305.
Fischer. WJ .. Altheimer. S.. Cattori. v .. Meier, P.J.. Dietrich. D.R .. Hagenbuch. B.. 2005.
cytotoxicity (Fig. 8) and MC-Western blotting of cell homogenates Organic anion transporting polypeptides ex pressed in liver and brain mediate
(Fig. 9). was reduced upon co-incubation of mWBC with the OATPI uptak e of microcystin. Toxicol. Appl. Pharmacol. 203. 257 - 263.
Oatp substrates TC and BSP. Both 50 tM BPS as well as 50 and 500 f1M Fischer. W] .. Dietrich. D.R .. 2000a. Pathological and biochemical characterization of
microcystin-Induced hepatopancreas and kidney damage in carp (Cyprinus caTpio).
TC reduced uptake of MC congeners. albeit TC with a much greater loxicol. Appl. Pharmacol. 164. 73- 81.
efficacy than BSP. Provided that the observed MC cytotoxicity is Fischer. W.J .. Hitzfeld. B.C.. Tencalla. F.. Eriksson. J.E .. Mikhailov. A.. Dietrich. D.R.. 2000b.
mediated via mOatp dependent transport. the observed differences in Microcystin-lR toxicodynamics. induced pathology. and immunohistoche mical
localization in livers of blue-green algae exposed rainbow trout (OTlcorh,VTlchus
reduction of MC -congener dependent cytotoxicity by BSP and TC may mykiss). Toxicol. Science 54. 365-373.
stem from varying affinities of tile three MC-congeners. TC and BSP for Fischer. w .J.. Dietrich. D.R.. 2000c. Toxicity of the cyanobacterial cyclic heptape ptide
the mOatp detected in the mWBC employed. However. only specific toxins microcystin-LR and -RR in early life-stages of the African clawed frog
(Xenopus laevis). Aquatic Toxicology 49. 189-198.
expression of these mOatp. e.g. in Xenopus oocytes (Fischer et al..
Fladmark. K.E.. Brustugun. O.T.. Mellgre n. G.. Krakstad. C.. Boe. R.. Vintermyr. O.K..
2005). HeLa (Monks et al.. 2007) or HEK293 cells (Komatsu et al.. Schulman. H.. Doskeland. S.o.. 2002. Ca2+/calmodulin-dependent protein kinase 11
2007) and subsequent kinetic analyses of MC-congener dependent is required for microcyst in-induced apoptosis. J. BioI. Chem. 277. 2804-28 11.
transport would allow deduction whether the mOatps in mWBC are Pu. WY.. Chen.J.P.. Wang. X.M .. Xu.l.H .. 2005. Altered expression ofp53. BcI-2 and Bax
induced by microcystin- LR in vivo and in vitro. Toxi.con. 46. 171 - 177.
primarily responsible for MC transport. Gao. B.. Hagenbuch. B.. Kullak-Ublick. G.A .. Benke. D.. Aguzzi. A.. Meier. P.J .. 2000.
The data of this study thus suggest that MC -LF and MC-LW have a Organic anion-transporting polypept,ides mediate transport of opioid peptide.s
much higher potential for inducing neurotoxicity in mice than MC-LR. across blood-brain barrier. J. Pharmacol. Exp. Ther. 294. 73 - 79.
Gao. B.. Hubor. KD .. Wenzel. A.. Vavricka. S. R.. Ismair. M.G .. Reme. C. Meier. P.J., 2005.
Consequently. based on the high similarities of mOatp with human localization of organic anion transporting polype ptides in the rat and human ciliary
OATP. a more thorough investigation of different MC congeners body epithelium. Exp. Eye Res. 80. 61-72.
appears important for proper assessment of microcystin toxicity. Gehringer. M.M .. 2004. Microcys tin-lR and okada,ic acid-induced cellular effects: a
dualistic response. FEBS Lett. 557. 1- 8.
including neurotoxicity. and cancer risk in humans. rather than relying Gilroy. DJ. Kauffman. K.w.. Hall. RA. Huang. X.. Chu. F.S .. 2000. Assessi ng potential
on MC-LR risk assessment (WHO. 1998). health risks from microcystin toxins in blue-green algae dietary su pplements.
Environmental Health Perspectives 108. 435- 439.
Hagenbuch. B.. Meier. I'.J.. 2003. The superfamily of organic anion transporting
Acknowledgments
polypeptides. Biot him. Biophys. Acta. 1609. 1- 18.
Hagenbuch. 8.. Meier. P.J .. 2004. Organic anion transporting polypeptides of the OATP/
This study was supported by the International Research Training SlC21 family: phylogenetic classi fication as OATP/SlCO superfamily. new nomen-
Group 1331 (IRTG 1331). We would like to thank. Or. Elisa May (Bio clature and molecular/functional properties. Pflugers. Arch. 447, 653-665.
Hastie. Cj.. Borthwick. E.B .. Morrison. l.F.. Codd. G.A.. Cohen. P.T.. 2005. Inhibition of
Imaging Centre. University of Konstanz. Germany) for the introduction several protein phosphatases by a non-covalenriy interacting microcystin and a
to con-focal microscopy. Prof. Dietrich Keppler (Division of Tumor novel cyanobacterial peptide. nostocyclin. Biochim. Blophys. Acta. 1726. 187- 193.
Biochemistry. German Cancer Research Centre. HeidelbeIg. Germany) Ito. E.. Kondo. F.. Harada. K.. 2000. First report on the d.istribution of orally administered
microcystin-LR in mouse tissue using an immunostaining method. Toxicon. 38.
for providing stably transfected HEK293-0ATPl B3 and Alexandra 37-48.
Heussner for the technical support Special thanks are due to Or. Jacquemin. E.. Hagenbuch. B.. Stieger. B.. Wolkoff. A.w.. Meier. P.J.. 1994. Express ion
Bernhard Ernst for his great support with the rPPIA assay and for his cloning of a rat liver Na( +)-i nde pendent organic anion transporter. Proc. Natl. Acad.
Sci. U. S. A 91. 133-137.
contributions to this project. The editorial review of the manuscript by Jochimsen. E.M.. Carmichael. WW.. An. J.5 .. [ardo. D.M.. Cookson. S.T.. Holmes. CE..
Dr. Evelyn O'Brien is gratefully acknowledged. Finally we would like to Antunes. M.B.. de Melo Filho. D.A .. Lyra. T.M .. Barreto. V.S .. Azevedo. S.M .. Jarvis. W.R..
thank Oliver Okle for the technical assistance. 1998. liver failure and death after exposure to microcystins at a hemodialysis center
in Brazil. N. Eng!.J. Med. 33 8. 873 - 878.
Kanai. N.. Lu. R.. Bao. Y.. Wolkoff. A.W. Sc huster. V.l.. 1996. Transient expression of oatp
References organic anion transporter in mammalian cells: identification of candidate
substrates. Am. J. Physiol. 270. F319- F325 .
Abe. T.. Kakyo. M.. Tokui. T.. Nakagomi . R.. Nishio. T.. Nakai. D.. Nomura. H.. Unno. M.. Komatsu. M.. Furukawa. T.. Ikeda. R.. Takumi. S.. Nong. Q.. Aoyama. K.. Akiyama. S..
Suzuki. M.. Nairoh. T.. Matsuno. S.. Yawo. H.. 1999. Identification of a novel gene Keppler. D.. Takeuchi. T.. 2007. Involvement of mitogen-activated protein kinase
family encoding human liver-specific organic anion transporter lST-1. J. BioI. Chem. signaling pathways in microcystin-lR-induced apoptosLS after its selective uptake
274. 17159-17163. mediated by OATPIBl and OATPtB3. Toxicol. Sci. 97. 407- 416.
Azevedo. S.M .. Carmichael. WW.. Jochim~en. E.M .. Rinehart. K.l.. lau. S.. Shaw. GX. and Kbnig.J .. Cui. Y.. Nies. AT.. Keppler. D.. 2000a. A novel human organic anion transporting
Eaglesham. G.K. (2002). Human intoxication by microcystins during renal dialysis polypeptide localized to the basolateral hepatocyte membrane. Am. J. Physiol.
treatment in Caruaru- Brazil. Toxicology 181 - 182. 441-446. Gastrointest. Liver Physiol. 278. GI56- GI64.
Batista. T.. de SOUSd. G.. Sup ut. J.5.. Rahm.tni. R.. Suput. D.. 2003. Microcystin-LR causes Kbnig. J.. Cui. Y.. Nies. A.T.. Keppler. D.. 200Gb. localization and genomic organization of
the collapse of actin filaments in primary human hepatocytes. Aquat. Toxico!. 65. a new hepatocellular organic anion transporting polypeptide. J. BioI. Chem. 275.
85- 91. 23161 - 23168.
 255

Kullak-Ublick, G,-A" Hagenbuch, B" Stieger, B" Wolkoff, A,W" Meier, P.j., 1994. Mosmann, l .. 1983, Rapid colorimetric assay for cellular growth and survival:
Functional characterization of the basolateral rat liver orga.nic anion transporting application to proliferation and cytotoxicity assays.j.lmmunol. Methods 65, 55-63,
polypeptide, Hepatology 20,411-416. Millhardt. C, 2002. Der Experimentator: Molekularbiologie. 3. Aufi .. Spektrum,
Kullak-Ublick, GA, Hagenbuch, H" Stieger, B" Schteingart, CD" Hofmann, A.f" Wolkoff, Heidelberg, Germany.
AW" Meier, P.j., 1995. Molecular and functional characteriZdtion of an organic anion Naud, j .. Michaud, j .. Boisver!, c..Desbiens, K.. Leblond, fA. Mitchell, A.. jones, C,
transporting polypeptide cloned from human liver. Gastroenterology 109, 1274- 1282. Bonnardeaux, A. Pichette. V.. 2007. Down-regulation of intestinal drug transpOlters
Kullak-Ublick, GA, Stieger, B" Meier, P.j., 2004. Enterohepatic bile salt transporters in in chronic renal failure in rats. j. Phamlacol. Exp. Ther. 320. 978- 985.
normal physiology and liver disease. Gastroenterolo&'Y 126, 322 - 342. Nies, AT.. 2007. The role of membrane transporters in drug delivery to brain tumors.
lawrence, j.F., Niedzwiadek, H" Menard, c.. lau, 8.1'., Lewis, D" Kuiper-Goodman, T" Cancer Let!. 254, 11 - 29.
Carbone, S" Holmes, c.. 2001. Comparison of liquid chromatography{mass Nishiwaki, R.. Ohra. T.. Eisaburo, S" Suganuma, M .. Harada, K.-!., Watanabe, M.F., Fujiki,
spectrometry, ELlSA, and phosphatase assay for the determination of microcystins H" 1994. Two significant aspects of microcystin-LR: specific binding and liver
in blue-green algae products, journal of the AOAC Intemational 84, 1035- 1044, specificity. Cancer Lett. 83, 283 - 289,
laemmli, u'K" 1970, Cleavage of structural proteins during the assembly of the head of Pouria, 5" de Andrade, A.. Barbosa, j .. Cavalcanti. RL, Barreto, V.T.. Ward, c.j" Preiser, Woo
bacteriophage T4. Nature 227,680-685, Poon, G.K" Neild, G.H .. Codd, G,A .. 1998. Fatal microcystin intoxication in
Lee, W" Glaeser, H" Smith, LH" Roberts, KL., Moeckel. G.w" Gervasini, G" Leake, B,E, haemodialysis unit in (aruaru, Brazil. lancet 352, 21 - 26.
Kim, R.B" 2005, Polymorph isms in human organic anion-transporting polypeptide Rozen, S" Skaletzky, H.j .. 2000. Primer3 on the WWW for general users and biologist
1A2 (OAll'IA2): implications for altered drug disposition and central nervous programmers. In : Krawetz. 5" Misener. S. (Eds.), Bioinformatic Methods and
system drug entry. j. Hiol. Chem. 280, 9610-9617. Protocols: Methods in Molecular Biology. Human Press, Totowa, Nj, pp, 365- 386.
Lu, H., Choudhuri, S" Ogura, K" Csanaky, I.L., Lei, X.. Cheng, X.. Song, P.Z.. Klaassen, CO .. Runnegar, M .. Berodt, N.. Kaplowitz, N.. 1995. Microcystin uptake and inhibition of
2008. Characterization of organic anion transporting polypeptide 1b2-null mice: protein phosphatases: effects of chemoprotectants and self-inhibition in relation to
essential role in hepatic uptake{toxicity of phalloidin ,lnd microcystin-LR. ·loxicol. known hepatic transporters. Toxicol. Appl. Pharmacol. 134, 264-272.
Sci. 103,35-45. Runnegar. M.T.. Gerdes, KG .. Falconer, !.R .. 1991. The uptake of the cyanobacterial
MacKintosh, c.. Beartie, K.A .. Klumpp, S" (ohen, P.. (odd, GA, 1990. Cyanobacterial hepatotoxin microcystin by isolated rat hepatocytes. Toxicon. 29,43-51.
microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A Sai, Y" Kaneko, Y" Ito, 5.. Mitsuoka, K.. Kato, Y.. Tamai, I., Artursson, p.. Tsuji, A.. 2006.
from both mammals and higher plants. FEBS Let!. 264, 187 - 192. Predominant contribution of organic anion transporting polypeptide OATI'-B
MacKintosh, C, MacKintosh, R.W" 1994. Inhibitors of protein kinases and phosphatases. (OATP2B I) to apical uptake of estrone-3-sulfate by human intestinal (aco-2 cells.
Trends in Biochemical Science 19. 444-448, Drug Metab, Dispos. 34, 1423-1431.
Mankiewicz, j" Tarczynska, M" f1admark, K.E.. Doskeland, S.O" Waiter, Z" Zalewski, M .. Schaeffer, 0" Malpas, p" Barton, L, 1999. Risk assessment of microcystin in dietary
2001. Apoptotic effect of cyanobacterial extract on rat hepatocytes and human Aphanizomenon jlos-aq,/Qe. Ecotoxicology and Environmental Safety 44, 73 - 80,
Iymphocytes. Environ. Toxicol. [print[16, 225- 233, Soares, R.M .. Yuan, M .. Servaites, jL Delgado, A" Magalhaes, V,F.. Hilbom, E,D"
Meier-Abt. F" Hammann-Hanni, A" Stieger, B.. Ballatori, N.. Boyer, j.L., 2007. The Carmichael. WW" Azevedo, S.M" 2006, Sublethal exposure from microcyst ins to
organic anion transport polypeptide I d I (Oatp ld I) mediates hepatocellular uptake renal insufficiency patients in Rio de janeiro, Brazil. Environ. Toxicol. 21. 95 - 103.
of phalloidin and microcystin into skate liver. Toxicol. Appl. Pharmacal. 218, Tani, T.. Gram, LK .. Arakawa, H.. Kikuchi, A., Chiba, M" Ishii, Y.. Steffansen. B" Tamai, I.. 2008,
274-279. Involvement of organic anion transporting polypeptide I a5 (Oatpl as ) in the intestrnal
Meriluoto,j.A.. Nygard, S.E.. Dahlem, A.M., Eriksson, j .E.. 1990. Synthesis. organotropism absorption of endothelin receptor antagonist in rats. Phanm. Res. 25, 1085- 1091.
and hepatocellular uptake of two tritium-Iabeled epimers of dihydromicrocystin- 'Iobwin, H.. Staehelin, T.. Gordon, j., 1979. Electrophoretic transfer of proteins from
LR, a cyanobacterial peptide toxin analog, Toxicon, 28, 1439-1446. polyacrylamide gels to their nitrocellulose sheet. Procedure and some applications.
Meriluoto, jA. Spoof, L.E.. 2008. (yanotoxins: sampling, sample processing and toxin Proc. Natl. Acad. Sci. USA 76, 4350- 4354.
uptake. Adv. Exp. Med. BioI. 619,483-499. Toivola, D.M .. Eriksson, j.E.. Brautigan, D.L, 1994. Identification of protein phosphatase
Mikhailov, A., Harmala-Brasken, A.-S .. Polosukhina , E" Hanski, A.. Wahlsten, M" 2A as the primary target for microcystin-LR in rat liver homogenates. FEBS Lett. 344,
Sivonen, K" Eriksson,j.E" 2001. Production and speci ficity of monoclonal antibodies 175-180.
against nodularin conjugated through N-methyldehydrobutyrine, Toxicon. 39, Weng, D.. Lu, Y" Wei, Y.. Liu, Y.. Shen, P., 2007, The role of ROS in microcystin-LR-induced
1453- 1459. hepatocyte apoptosis and liver injury in mice. Toxicology 232, 15- 23,
Mikkaichi. T.. Suzuki. T.. Tanemoto, M., Ito, S" Abe, T" 2004. The organic anion WHO, 1998. Cyanobacterial toxins: microcystin-LR. Guidelines for Drinking-Water
transpOlter (OATP) family, Drug Metab. Pharmacokinet. 19, 171 - 179. Quality, World Health Organizat.ion, Geneva, pp. 95-110.
Monks, N,R.. Liu, S" Xu, Y.. Vu, H.. Bendelow, A.S., Moscow, j.A" 2007, Potent cytotoxicity Wickstrom. M.l.. Khan, SA, Haschek, WM" Wyman, j.F., Eriksson, j.E., Schaeffer, DJ..
of the phosphatase inhibitor microcystin LR and microcystin analogues in Beas ley, V.K. 1995, Alterations in microtubules, intermediate filaments, and micro-
OATI' 1B1- and OATI'I B3-expressing Hela cells. Mol. Cancer Ther. 6, 587-598, filaments induced by microcystin-LR in cultured cells. Toxical. Pathol. 23, 326 - 337,