Supplemental information

Tab. 4.1 (S): Ratios of PP1 and PP2A versus GAPDH in HEK293 transfectants and primary human hepatocytes following densitometric evalutation of the western blots.

Density PP1/GAPDH PP2A/GAPDH

HEK293-CV 1,12 1,11

HEK293-OATP1B1 1,08 1,33

HEK293-OATP1B3 1,19 1,27

human hepatocytes 0,35 0,08

Mean PP1/GAPDH PP2A/GAPDH

HEK293-CV 1,12 1,11

HEK293-OATP1B1 1,08 1,32

HEK293-OATP1B3 1,19 1,27

human hepatocytes 0,35 0,08

Volume PP1/GAPDH PP2A/GAPDH

HEK293-CV 1,12 1,11

HEK293-OATP1B1 1,08 1,33

HEK293-OATP1B3 1,19 1,27

human hepatocytes 0,35 0,08

Chapter IV OATP-mediated MC Congener Toxicity

Fig. 4.1 (S): Immunoblot detection of PP1 and PP2A in primary human hepatocytes (mixture between donor 1 and 2), HEK293 cells transfected with a control vector (CV), OATP1B1 and OATP1B3. Equal amounts of total protein (60 µg/lane) were applied.

Molecular weights were estimated by comparison with marker proteins. Due to limited availability of primary human hepatocytes the detection could not be normalized against a housekeeping protein.

Acknowledgements

We would like to acknowledge the Arthur & Aenne Feindt Foundation (Hamburg, Germany), the BMBF (01GG0732-AKN), as well as the European Union (PEPCY QLRT-2001-02634) for kindly funding parts of this study. We would also like to thank Prof. Dr. Dietrich Keppler (Division of Tumor Biochemistry, German Cancer Research Centre, Heidelberg, Germany) for kindly providing the transfected HEK293 cells, Dr. Elisa May, Christine Strasser and Daniela Hermann (Bio Imaging Centre, University of Konstanz, Germany) for the introduction and assistance to laser confocal microscopy, as well as Alicja Panas, Jasmin Leyhausen, Julia Kleinteich and Isabelle Eisele for practical assistance.

primary human hepatocytes HEK293-CV

HEK293-OATP1B1

HEK293-OATP1B3 PP1

PP2A

primary human hepatocytes HEK293-CV

HEK293-OATP1B1

HEK293-OATP1B3 PP1

PP2A

5 In vitro toxicity of cylindrospermopsin in primary human hepatocytes and OATP-expressing HEK293 cells

Fischer A¹, Feurstein D¹, Knobeloch D², Nussler A³, Dietrich DR¹

1 Human and Environmental Toxicology, University of Konstanz, Konstanz, Germany

2 Department of General, Visceral, and Transplant Surgery, Charité Campus Virchow, Berlin, Germany

3 Technical University Munich, Department of Traumatology, Munich, Germany

in preparation

5.1 Abstract

The cyto- and genotoxic cyanobacterial alkaloid cylindrospermopsin (CYN) is presumed to be the causative toxin for the severe cases of hepatoenteritis on Palm Island in 1979. In vivo studies revealed the liver to be the organ predominantly affected following exposure to CYN. Its toxicity was demonstrated to stem from the inhibition of protein synthesis at low concentrations (maximum inhibition at 0.5 µM) and P450-mediated cytotoxicity at high concentrations (1-5 µM) using primary mouse hepatocytes. Despite the hydrophilic character of CYN its low molecular weight of only 415 Da might enable passive diffusion. However, evidence for the involvement of the bile acid transport system has been given by in vitro studies using primary rat hepatocytes.

Therefore, we compared the cytotoxicities of CYN with OATP transport-dependent MCLR and cell-permeant OA in primary human hepatocytes of two different donors at 48 h and liver OATP1B1- and OATP1B3-transfected, as well as mock-transfected HEK293 cells at 48, 72 and 96 h using the MTT reduction assay. In primary human hepatocytes CYN, MCLR and OA elicited typical dose-response effects on viability with 48-h EC50s ranging from 271.3 - 324.2 nM,

Chapter V CYN Uptake and Toxicity

3.42 - 24.63 nM and 3.01 - 35.05 nM, respectively. On the contrary, in OATP-expressing HEK293 cells we estimated a time-independent EC50 value of approximately 5000 nM (highest concentration applied), whereas viability in mock-transfected HEK293 cells remained above 50%. This supports the involvement of liver OATPs in CYN uptake, even though OATP-mediated CYN toxicity was not as evident as for MCLR. In addition, coincubation of CYN with OATP substrates bromosulphophthalein and taurocholate failed to protect HEK293 cells against CYN toxicity. On the other hand, CYN appeared to protect OATP-expressing HEK293 cells against toxic concentrations of BSP.

The results suggest some involvement of OATPs in transporting CYN, but further investigations are needed to clearify their importance and contribution to CYN toxicity.

Keywords: cyanobacteria, cylindrospermopsin, primary human hepatocytes, OATP

5.2 Introduction

In 1992 Ohtani et al. (Ohtani et al., 1992) identified cylindrospermopsin (CYN) from its eponymous producer Cylindrospermopsis raciborskii which is considered as the causative organism of the Palm Island Mystery Disease, a major outbreak of severe hepatoenteritis on Palm Island near Queensland, Australia in 1979 (Bourke et al., 1983; Hawkins et al., 1985). CYN is a cyanobacterial alkaloid with a molecular weight of 415 Da. It is a highly water-soluble zwitterion that is composed of a sulphated and methylated tricyclic ring structure containing a guanidine moiety that is linked to a hydroxymethyluracil (Ohtani et al., 1992; Falconer, 2005a). To date only two structural variants, 7-epicylindrospermopsin (Banker et al., 2000) and 7-deoxycylindrospermopsin (Norris et al., 1999) have been found to occur naturally.

CYN predominantly targets the liver, causing dose-dependent necrosis and haemorrhages, however, other organs, such as kidneys, lungs, thymus, heart, stomach and small intestine were found to be affected as well, as demonstrated by various exposure studies in which mice were treated either with extracts of

(Hawkins et al., 1985; Ohtani et al., 1992; Terao et al., 1994; Hawkins et al., 1997; Falconer et al., 1999; Seawright et al., 1999). For the purified toxin i.p.

LD50 values of 2100 and 200 µg/kg bw were determined at 24 hours and 5 - 6 days, respectively (Ohtani et al., 1992). A provisional guidline value of 1 µg/l for drinking water has been suggested based on no-observed-adverse-effect levels (NOAELs) and several uncertainty factors (Shaw et al., 2000; Humpage and Falconer, 2003). The toxicity of CYN relies on the inhibition of protein biosynthesis as shown in reticulocyte lysates (Terao et al., 1994; Froscio et al., 2001; Runnegar et al., 2002) and primary rat hepatocytes, in which inhibition of GSH synthesis was additionally reported (Runnegar et al., 1995d; Runnegar et al., 2002). Moreover, evidence for the involvement of CYN metabolization by cytochrome P450 enzymes (Terao et al., 1994) that generate (an) active metabolite(s) have been provided in vitro (Runnegar et al., 1995d; Froscio et al., 2003; Humpage et al., 2005) and in vivo (Norris et al., 2002). In fact, Froscio et al. (Froscio et al., 2003) distinguished between two events in CYN toxicity using primary mouse hepatocytes: the inhibition of protein synthesis that appeared to be irreversible and an early response at lower concentrations (maximum inhibition at 0.5 µM) and P450-mediated cytotoxicity at acute concentrations (1-5 µM). In addition, studies on DNA alteration provided evidence of CYN to be genotoxic forming DNA adducts (Shaw et al., 2000) and causing strandbreaks in hepatocytes of CYN treated mice (Shen et al., 2002), as well as a loss of whole chromosomes in a human lymphoblastoid cell line (Humpage et al., 2000;

Humpage et al., 2005). Fessard et al. (Fessard and Bernard, 2003) suggested an active CYN metabolite as being responsible for the reported genotoxicity, since strandbreaks failed to appear in CHO-K1 cells in which metabolizing enzyme activities are low. Indeed, inhibitors of CYP450 prevented CYN induced DNA fragmentation in primary rat hepatocytes (Humpage et al., 2005).

In general, permanent cell lines appeared to be less susceptible to CYN than primary hepatocytes (Shaw et al., 2000; Chong et al., 2002). This might be due to a lower CYP450 activity in the cell lines. E.g. although a decrease in mRNA contents of most CYPs has been reported in primary hepatocytes after 24 hours of culturing, enzyme activities retained and levels were still 400 times higher than in hepatoma cells (Rodriguez-Antona et al., 2002). In addition, reduced susceptibility might also stem from a lack of membrane transporters, as it is

Chapter V CYN Uptake and Toxicity

commonly observed in cell lines derived for example from liver and gut like McArdle RH-7777 (Torchia et al., 1996) and HepG2 (Marchegiano et al., 1992;

Torchia et al., 1996; Boaru et al., 2006) that might mediate the uptake of CYN.

Due to the hydrophilic character of CYN, Runnegar et al. (Runnegar et al., 2002) considered passive diffusion through cell membranes unlikely, suggesting the requirement of transporter-mediated uptake into cells and members of the solute-carrier family as possible candidates. However, they found that the inhibtion of protein synthesis was independent of transport in vitro. Chong et al.

(Chong et al., 2002) demonstrated a dose-dependent increase in viability of primary hepatocytes exposed to lethal concentrations of CYN (800 ng/ml) at 48 hours when coincubating with the bile acids, cholate and taurocholate. In addition, the authors reported that KB cells devoid of the bile acid transport system were approximately 8 times less susceptible to CYN than the primary hepatocytes and suggested passive diffusion or further transport systems as additional uptake mechanisms.

To our knowledge no experiments on CYN toxicity and uptake into human cells have been conducted. Therefore, the purpose of this study was 1.) to determine CYN cytotoxicity in primary human hepatocytes and 2.) to asses the potential role of human liver OATPs on the uptake of CYN by comparing its cytotoxic effects in HEK293 cells stably expressing OATP1B1 and OATP1B3 in the presence or absence of the known OATP substrates taurocholate (TC) and bromosulphophthalein (BSP) (Meier and Stieger, 2002; Hagenbuch and Meier, 2003; Hagenbuch and Meier, 2004). Both substrates have already been used to competitively inhibit the uptake of MCLR, a cyanobacterial serine/threonine-specific protein phosphatase inhibitor (Honkanen et al., 1990; MacKintosh et al.;

MacKintosh, 1993; Runnegar et al., 1993; Runnegar et al., 1995a; Hastie et al., 2005), by different OATPs (Fischer et al., 2005; Komatsu et al., 2007; Monks et al., 2007; Feurstein et al., 2009; Fischer et al., 2010).

The experiments were carried out with MCLR and OA, a cell-permeant serine/threonine-specific protein phosphatase inhibitor produced by different genera of dinoflagellates (Haystead et al., 1989; Cohen et al., 1990; Hardie, 1993), as control for OATP-mediated uptake and transporter-independent uptake, respectively.

5.3 Material & Methods

Chemicals and reagents

All chemicals were of the highest analytical grade commercially available.

Standards of cylindrospermopsin (CYN), microcystin-LR (MCLR) and okadaic acid (OA) were obtained from Alexis (Switzerland) and Sigma (Germany), respectively. Dried purified CYN was additionally provided by Dr. Andrew R.

Humpage (Department of Clinical and Experimental Pharmacology, University of Adelaide, Adelaide, Australia). CYN was dissolved in pure water, diluted to the stock and working concentrations according to the providers’ specifications.

Concentrations, stability and purity were repeatedly confirmed at the beginning and throughout the experiments by reversed-phase HPLC-DAD. The analysis was carried out according to the method of Lawton et al. (Lawton et al., 1994) for the analysis of MCs, however, with detection performed at a wavelength of 262 nm. A system of Shimadzu consisting of a Shimadzu system controler SCL-10Avp, Shimadzu auto injector SIL-10Advp, two Shimadzu pumps LC-10ATvp, Shimadzu diode array detector SPD-M10Avp, Shimadzu degasser DGU-14A and a Grom C18 reversed-phase column Grom-Sil 120 ODS-4 HE, 4.6 x 250 mm, 5 µm particle size was employed. MCLR was dissolved in 75% MeOH. The concentrations of the stock and the working solutions were confirmed photometrically using the molar absorption coefficient of MCLR (39800 mol l^-1 cm^-1) published by Harada et al. (Harada et al., 1990a). This coefficient refers to MCLR dissolved in 100% MeOH, however, was shown to be applicable for 75%

MeOH as well (Meriluoto et al., 2004; Meriluoto and Spoof, 2005). Additionally, MCLR concentrations were confirmed by HPLC-DAD analysis according to Lawton et al. (Lawton et al., 1994) using the same system described for CYN analysis. Okadaic acid (OA) (Sigma, Germany) was dissolved in 100% H2O, diluted to the stock and working concentrations according to the manufacturer’s specifications. All toxins were sterilized by filtration using a 0.22 µm filter (Millex-GV, sterile; Millipore, Ireland).

Cell systems

Human primary hepatocytes were isolated in agreement with the ethical review board and after the patients written consent by a standard operating procedure

Chapter V CYN Uptake and Toxicity

and cultured as described previously (Nussler et al., 2009). Both donors were females. Donor 1: partial liver resection due to cholangiocarcinoma (CCC), born 1961 and donor 2: partial liver resection due to liver metastasis of primary mamma carcinoma, born 1965.

Human embryonic kidney cells (HEK293) stably transfected with recombinant human organic anion transporting polypeptides 1B1 (HEK293-OATP1B1) and 1B3 (HEK293-OATP1B3), as well as a control vector (HEK293-CV) were kindly provided by Prof. Dietrich Keppler (Division of Tumor Biochemistry, German Cancer Research Centre, Heidelberg, Germany). HEK293 cells were cultured as described in chapter IV.

CYN cytotoxicity in primary human hepatocytes and OATP- and control vector-transfected HEK293cells

For cytotoxicity experiments human primary hepatocytes of two different donors were sent seeded in collagen-coated 96-well plates (Price, 1975) at a density of 3 x 10⁵ cells/ml and 200 µl/well (6 x 10⁴ cells/well). Subsequent to arrival the cells were incubated at 37°C and 5% CO2 for 4-5 h. Prior to exposure the medium was decanted and replaced by 200 µl RPMI 1640 containing 100 units/ml penicillin and 100 mg/ml streptomycin. Passages 3-8 of transfected HEK293 cells were used for toxin exposure experiments. HEK293 cells were seeded in 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany), coated with poly-L-lysine (5 mg/ml), in MEM (supplemented as described above) at the same density as hepatocytes. After 4-5 h the medium was decanted and cells were incubated in 200 µl MEM (supplemented as described above, but with 1% FBS). Primary hepatocytes and HEK293 cells were incubated with CYN serially diluted (1:3) from 5000 nM to 2.29 nM for 48 h and 48, 72 and 96 h, respectively. HEK293 cells were additionally exposed to MCLR and OA in serial dilutions (1/3) from 5000 nM to 2.29 nM and 100 nM to 0.003 nM, respectively. Cytotoxicity data of MCLR and OA on primary human hepatocytes have been collected alongside with CYN, but will be published elsewhere (Fischer et al., 2010).

Coincubation studies on CYN with bromosulfophthalein and taurocholate

Coincubation studies of CYN and taurocholate (TC) or bromosulfophthalein (BSP) on HEK293 cells were performed under identical conditions as described for single toxin exposures. Cells were exposed 4-5 h after seeding to 5 µM CYN coincubated with concentrations of 500 µM, 50 µM and 5 µM TC and 100 µM, 50 µM and 5 µM BSP, respectively, for 72 h and 96 h. As controls and for comparison, cells were additionally exposed to CYN and both OATP substrates without coincubation at the following concentrations: CYN serially diluted (1:3) from 5000 nM to 2.29 nM, BSP at 100 µM and 33.3 µM and TC at 500 µM and 166.7 µM.

MTT reduction assay

Following exposure of HEK293 cells or human hepatocytes, 20 µl MTT (5 mg/ml) solution was added to each well and cells were incubated for 1.5 h.

Subsequently the medium was removed carefully by pipette. The formed insoluble MTT-formazan was redissolved by adding 100 µl solubilization buffer (95% (v/v) isopropanol, 5% (v/v) formic acid) to each well and gentle shaking of the plate for at least 15 min. Finally, absorption was measured at 550 nm in a microtitre plate reader (Tecan, microplate reader, infinite M200; Austria). As positive control (0% survival) cells were incubated with 1% TWEEN (8 wells/96-well plate). The highest MeOH and water concentrations in the assays were

<2% and 12.5%, respectively, and were used as the corresponding solvent controls (8 wells/96-well plate). The solvent controls served as negative controls (100% survival) and were compared to cells incubated with medium only (8 wells/96-well plate). No differences in viability, condition or growth rate could be identified between solvent and negative control (data not shown).

Statistics

Cytotoxicity studies were carried out at least three times in duplicates. The mean values of each duplicate yielded the values for calculation of the standard deviation (n≥3). For calculation of the respective R², EC50 values and statistical analysis GraphPad Prism 4.03 software was used. Briefly, the respective mean values were log-transformed and normalized. The resulting curves were fitted by nonlinear regression. An F-test (P<0.05) was employed for for the comparison of the EC50 values, hillslopes and curves. One-way ANOVA

Chapter V CYN Uptake and Toxicity

followed by Tukey's Multiple Comparison Test was employed to compare the effects of the highest concentration of CYN (5 µM) on transfected HEK293 cells at the respective time of exposure.

Coincubation studies were performed at least three times in duplicates. The mean values of each duplicate yielded the values for calculation of the standard deviation (n≥3). Data were statistically analyzed by One-way ANOVA followed by Dunnett’s Multiple Comparison Test, whereas the respective negative control (medium control) was used as reference. One-way ANOVA followed by Tukey's Multiple Comparison Test was employed to assess significant differences between the data of the coincubation studies.

5.4 Results

Cytotoxicity of CYN in primary human hepatocytes and liver OATP-expressing HEK293 cells

CYN elicited typical concentration-response effects in primary human hepatocytes (Tab. 5.1; Fig. 5.1), however, with donor-dependent differences in susceptibility resulting in 48 h EC50 values of 271.3 nM (donor 1) and 324.2 nM (donor 2). In general, donor 1 appeared to be more susceptible showing increased sensitivity towards MCLR and OA (see chapter IV; Tab. 4.1) as well.

HEK293 cells appeared to be far less susceptible towards CYN than hepatocytes (Tab. 5.1; Fig. 5.2 (A-C)). After 48 h a decrease of approximately 50% in viability was only measured in HEK293-OATP1B3 at the highest CYN concentration (5 µM).

Tab. 5.1:48 h EC50 values with 95% confidence intervals in brackets of CYN, MCLR and OA in primary human hepatocytes.

*, Fischer et al., 2010

Fig. 5.1: 48 h cytotoxicity of CYN in primary human hepatocytes of two different donors. Negative control = solvent control. Values represent mean ± standard error of the mean of at least three independent experiments.

primary human hepatocytes

(Donor 1)

primary human hepatocytes

(Donor 2)

CYN 271.3 nM

(245.5 - 299.9) R² = 0.9869

324.2 nM (237.1 - 443.4)

R² = 0.9062

MCLR *3.4 nM

(2.85 - 4.12) R² = 0.9647

*24.6 nM (17.45 - 34.78)

R² = 0.9320

OA *3.0 nM

(2.41 - 3.77) R² = 0.9698

*35.1 nM (24.46 - 50.21)

R² = 0.8394)

10^-1 10⁰ 10¹ 10² 10³ 10⁴ 0

25 50 75 100

125 Donor I

Donor II

CYN [nM]

viability [% negative control]

Chapter V CYN Uptake and Toxicity

Tab. 5.2:48, 72 and 96 h EC50 values with 95% confidence intervals in brackets of CYN, MCLR and OA in primary human hepatocytes and transfected HEK293 cells.

n.d., not determinable

HEK293-CV HEK293-OATP1B1 HEK293-OATP1B3

48 h 72 h 96 h 48 h 72 h 96 h 48 h 72 h 96 h

This value was reached by HEK293-OATP1B1 not until 72 h, whereas viability of HEK293-CV remained above 50% and at the same level during any time of exposure. However, statistical analysis revealed no significant time dependency of CYN toxicity in any of the transfected HEK293 cells (P>0.05; Tukey's Multiple Comparison Test).

Fig. 5.2: Cytotoxicity of CYN in HEK293-CV (A), HEK293-OATP1B1 (B) and HEK293-OATP1B3 (C) at 48, 72 and 96 h. Negative control = solvent control. Values represent mean ± standard error of the mean of at least five independent experiments.

On the contrary, MCLR cytotoxicity was concentration- and time-dependent in both OATP-expressing HEK293 cells (Tab. 5.2; Fig. 5.3 (B and C)): 48, 72 and 96 h EC50 values of 187.5 nM, 105.9 nM and 65.41 nM and 156.7 nM, 123.7 nM and 95.72 nM were calculated for HEK293-OATP1B1 and HEK293-OATP1B3, respectively. However, differences in the EC50s of HEK293-OATP1B3 were statistically insignificant (P>0.05; F-test), hence, data analysis resulted in a

10⁰ 10¹ 10² 10³ 10⁴

Chapter V CYN Uptake and Toxicity

single curve (Fig. 5.3 (C)). HEK293-CV remained insusceptible towards MCLR at any concentration and time of exposure (Tab. 5.2; Fig. 5.3 (A)).

Cell-permeant OA caused concentration-dependent effects in all HEK293 transfectants (Tab. 5.2; Fig. 5.4 (A-C)) with comparable EC50s in the low nanomolar range. However, the respective EC50 values indicate no time dependency of OA cytotoxicity: in HEK293-CV 48, 72 and 96 h EC50s were 9.41 nM, 11.65 nM and 9.25 nM, in HEK293-OATP1B1 7.02 nM, 9.09 nM and 6.27 nM and in HEK293-OATP1B3 4.70 nM, 7.08 nM and 5.56 nM, respectively.

Fig. 5.3: Cytotoxicity of MCLR in HEK293-CV (A), HEK293-OATP1B1 (B) and HEK293-OATP1B3 (C) at 48, 72 and 96 h. Negative control = solvent control. Values represent mean ± standard error of the mean of at least four independent experiments.

10⁰ 10¹ 10² 10³ 10⁴

Fig. 5.4: Cytotoxicity of OA in HEK293-CV (A), HEK293-OATP1B1 (B) and HEK293-OATP1B3 (C) at 48, 72 and 96 h. Negative control = solvent control. Values represent mean ± standard error of the mean of at least four independent experiments.

Coincubation studies on CYN with bromosulfophthalein and taurocholate using OATP- and control vector-transfected HEK293cells

For coincubation studies HEK293 cells were exposed only for 72 and 96 h to reduce sample size and improve menageability. Moreover, cytotoxicity data suggested differences to be rather significant at longer times of exposure.

However, neither TC nor BSP lead to a significant protection against CYN toxicity at 72 and 96 h in any HEK293 transfectant (Fig. 5.5 (A-F) and Fig.5.6 (A-F); P>0.05; Tukey's Multiple Comparison Test). Contrarily, 5 µM CYN elicited significant effects only in OATP-expressing HEK293 cells, whereas reduction of viability of HEK293-CV was insignificant (Fig. 5.5 (A and B) and Fig. 5.6 (A and B)).

Chapter V CYN Uptake and Toxicity

Fig. 5.5: Coincubation study (72 h). Cytotoxic effects in HEK293-CV (A and B), HEK293-OATP1B1 (C and D) and HEK293-OATP1B3 (E and F) following coincubation of CYN with TC or BSP. Negative control = solvent control. Values represent mean ± standard error of the mean of three independent experiments. Each

negativ

Fig. 5.6: Coincubation study (96 h). Cytotoxic effects in HEK293-CV (A and B), HEK293-OATP1B1 (C and D) and HEK293-OATP1B3 (E and F) following coincubation of CYN with TC or BSP. Negative control = solvent control. Values represent mean ± standard error of the mean of three independent experiments. Each respective negative control was used as reference for comparison (One-way ANOVA followed by Dunnet’s Multiple Comparison Test; * P<0.05; ** P<0.01).

negative control

Chapter V CYN Uptake and Toxicity

In addition, similar to the results obtained from CYN exposure experiments on HEK293 cells (Fig. 5.2 (A-C)) no distinct time dependency was observed within the coincubation studies.

Furthermore, high concentrations of BSP (100 µM) significantly reduced viability of OATP-expressing HEK293 cells at 72 and 96 h, whereas HEK293-CV remained unaffected. This reduction was diminished by coincubation with 5 µM CYN in both OATP-expressing HEK293 cells at 72 and 96 h (Fig. 5.5 (D and F) and Fig. 5.6 (D and F)). While this effect appeared to be independent of the

Furthermore, high concentrations of BSP (100 µM) significantly reduced viability of OATP-expressing HEK293 cells at 72 and 96 h, whereas HEK293-CV remained unaffected. This reduction was diminished by coincubation with 5 µM CYN in both OATP-expressing HEK293 cells at 72 and 96 h (Fig. 5.5 (D and F) and Fig. 5.6 (D and F)). While this effect appeared to be independent of the

Im Dokument In vitro investigations on uptake and toxicity of cyanobacterial toxins (Seite 70-91)