The uptake of the cyanobacterial hepatotoxin microcystin by isolated rat hepatocytes

Toxlcoe Vol . 29, No. 1, pp. 451, 1991 . Printed in Grut Btitaio.

0041-0IOIj91 53 .00 + .00 (%~ 1990 Pergemon Press pk

THE UPTAKE OF THE CYANOBACTERIAL HEPATOTOXIN MICROCYSTIN BY ISOLATED RAT HEPATOCYTES MARIA T . C . RUNNEGAR, * ROBERT G. GERDES

and

IAN R . FALCONER

Department of Biochemistry, Microbiology, and Nutrition, University of New England, Armidale, NSW, Australia (Accepted for publication 21 June 1990) M . T . C. RUNNEGAR, R . G . GERDES and I . R . FALCONER . The uptake of the cyanobacterial hepatotoxin microcystin by isolated rat hepatocytes. Toxicon 29, 43-51, 1991 .-Microcystin-YM a cyclic heptapeptide hepatotoxin isolated from the cyanobacterium Microcystis aeruginosa was radiolabeled with 'zSI, and used to investigate the uptake of the toxin by freshly isolated rat hepatocytes. The uptake was temperature dependent with apparent activation energy of 18 kcal/mole (77 kJ/mole) for the initial rate of uptake . Uptake of non-toxic (10-20 nM) doses of microcystin by hepatocytes continued with time, the intracellular to extracellular distribution ratio for the toxin was 70 at 60 min for 106 cells/ml . Uptake of higher doses of microcystin (100 nM and more) stopped when the cells blebbed: a toxic response of hepatocytes to microcystin. Uptake of microcystin by hepatocytes was inhibited 70-80% by the addition of 10 ~M sodium deoxycholate or bromsulphthlein, compounds that protect hepatocytes from the toxic effects of microcystin.

INTRODUCTION

are a family of potent hepatotoxic peptides (LDsp i.p. in mice 50-100 hg/kg) produced by the cyanobacterium Microcysti%s aeruginosa (BOTES et al., 1985; KRISxNAMURTI-Ix et al., 1989). Intoxication in vivo by these compounds is characterized by widespread hepatic necrosis and haemorrhage in a large number of species (JACKSON et al., 1985 ; HOOSER et al., 1989). We have shown that the heptapeptide microcystin-YM (cycloAla-Tyr-Measp-Met-Adda~lu-MeDha where Adda is 3-amino-9-methoxy2,6,8,-trimethyl-l0-phenyldeca-4,6-dienoic acid) can be easily iodinated with ' Z SI . In vivo the radioactively labeled peptide retains biological activity, and its toxicity cannot be distinguished from that of the native toxin (RUNNEGAR et al., 1986). Freshly isolated rat hepatocytes in suspension are used as an in vitro system as one method for the investigation of microcystin toxicity . Microcystin at 50-100 nM concentration causes rapid blebbing of hepatocytes, decreases in GSH content, activation of phosphorylase a (RUNNEGAR et al., 1987), increases in cytoplasmic levels of calcium as MICROCYSTTNS

'Present address and author to whom correspondence should be addressed : Division of Gastrointestinal and Liver Diseases, School of Medicine, University of Southern California, 2025 Zonal Ave, LAC 11-221, Los Angeles, CA 90033, U.S .A . 43

44

M. T. C. RUNNEGAR et al.

measured with Quin-2 and Fura-2 (FALCONER and RUNNEGAR, 1987) . These effects are early signs of injury to the cells that can be prevented by micromolar amounts of a number of substances such as bile acids (RUNNEGAR et al., ] 981) . The toxic effects are limited to hepatocytes, other cells tested so far have been shown to be insensitive to microcystin in the same dose range (FALCONER and RUNNEGAR, 1987; ERIKSSON et al., 1987) .

Biotransformation of compounds including xenobiotics and naturally occurring organics takes place in the liver. It is generally accepted that a large number of different compounds enter the liver by carrier mediated transport into hepatocytes. However, the nature and specificity of individual transport systems is in some cases controversial, with conflicting results and/or interpretations arising from different studies (FRIMMER and ZIEGLER, 1988) . In this study we have used '~I-labeled microcystin-YM and rat hepatocytes to characterize the uptake of microcystin by these cells. MATERIALS AND METHODS Microcystin-YM was purified from a Microcystis aeruginosa bloom from Malpas Dam (Armidale, Australia) by solvent extraction, gel filtration and high pressure liquid chromatography (BOTPS et al., 1985). Iodinated microcystin-YM was prepared from microcystin-YM by iodination with ['='n-sodium iodide (Amersham International, Sydney, Australia) by the lactoperoxidase (Sigma Chemical Co ., St Louis, MO, U.S .A .)/H,O, method (RuxxECATa et al., 1986; FALCOrtEa et al., 1986). Non radioactive iodinated ['nI]-microcystin was prepared in the same way as the radioactive derivative, except that ['nI]-sodium iodide was used. Native, monoiodinated and di-iodinated toxins were separated by reverse phase HPLC as described in RuxxECATt et al. (1986). The concentration of native and iodinated microcystin solutions was determined by measuring the absorbance at 240 nm (RUNNECAIt et al ., 1986). Hepatocytes were prepared from fed male Sprague-Dawley rats by collagenase perfusion (MOLDEUS et al., 1978). After an initial equilibration at 37°C for 30 min with shaking and continuous gassing with carbogen (95% O, : 5% COQ the cells in Krebs-Henseleit buffer pH 7.4 containing 12 .6 mM HEPES and 6 mM glucose had a viability (measured by Trypan blue exclusion) of at least 86%. Blebbing of hepatocytes by microcystin was determined by microscopic examination of incubation samples to which equal volumes of 10% formal-saline were added (RuxxrcAa er al ., 1981). For scanning electron microscopy, hepatocytes were fixed in 2% v/v glutaraldehyde in 0.1 M sodium phosphate buffer pH 7.2 and processed as described in RUNNEGAR et al. (1981) . Phosphorylase a activity was assayed according to the method of Hue et al. (1975) and MooaE et al. (1985) with minor modifications (RuxxeaATS et al ., 1987). Uptake studies of ["'I]-microcystin by hepatocytes were carried out in the same Krebs-Henseleit buffer at 30°C (unless otherwise stated) with shaking and carbogen gassing. Isolated hepatocytes (10 ml of 1 x 10° cells/ml) were incubated with a constant concentration of ['ul]-microcystin (3 .0-3 .5 x 10"counts/min/lOml) and varying concentrations of native microcystin-YM as shown in the results. To determine the time course of the uptake, aliquots of 500 pl of cell suspension were withdrawn, and the uptake was terminated by pelleting the cells by rapid centrifugation through a 500 pl layer of silicon oil of density 1 .041 g/liter (mixed Dow Corning DCSSO:DC200 in 7:l ratio, Ajax, Sydney, Australia) in a microfuge (BnTaTU1-r et al., 19ßl). Total radioactivity, and radioactivity associated with the cell pellet and/or the supernatant was measured in an automatic well-type scintillation counter (Packard Instrument Co., IL, U.S.A .) . To determine initial rates of uptake (v~ incubations were sampled every 15 sec, initial rates of uptake were calculated for the time intervals 30-b0 sec or 30-90 sec since for these time intervals the uptake was linear at all microcystin concentrations of 600 nM or less. lopodate, iodipamide were kind of gifts of Dr PErztxcEtt; antanamide was kindly given to us by Dr FAULSTICH.

RESULTS

t1sIJ-microcystin uptake: effects of dose and time Figure IA shows the cumulative uptake curve obtained when 1 x 106 hepatocytes/ml were incubated at 30°C with 17 nM ['ssI]-microcystin . The slope of the curve was maximal and linear over approximately 120 sec; the initial rate of uptake (v;) was taken to be the slope of this linear portion of the curve (see also Fig. 3). As time progressed the rate of uptake decreased, but significant amounts of microcystin continued to be taken up for ~

Microcystin Uptake by Hepatocytes

45

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so

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3o ao Time min

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60

~o

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FiG. 1 . CUMULATIVE UPTAx'E OF fzsn-MICROCYSTIN HY RAT i~PATOCYTES . Cells (I x 10°/ml) were incubated at 30°C with (A) 17 nM, (B) 417 nM ['al}microcystin and native microcystin. The incubations were sampled (0 .5 ml) at the times shown, after rapid centrifugation ; cell associated microcystin was determined by measuring the radioactivity of the cell pellet . The total radioactivity of each incubation was obtained by counting 0.5 ml samples. From these two values the actual amount of microcystin taken up at any given time point was calculated . Results are expressed as meant S.E .M . (n = 3) .

60 min.

By this time more than a third of the microcystin was cell associated. With increased concentrations of microcystin the cumulative uptake curve changed (Fig. 1 B). Uptake did not continue with time as in Fig. 1 A, so that at 417 nM toxin it ceased after about 10 min and was followed by a slight decrease in cell associated microcystin. On microscopic examination, the cessation of uptake coincided with blebbing of the hepatocytes. With increasing concentrations of microcystin the initial rate of uptake (v~ increased linearly without evidence of saturation up to 519 nM toxin concentration (Fig. 2). Attempts were made to measure uptake by hepatocytes of higher microcystin concentrations (2~ pM). In these experiments, in contrast to the findings of Fig. 2, the initial rate of uptake v; that was measured was significantly (P < 0.05) less than the value calculated by extrapolating Fig. 2 to higher microcystin concentrations . Nevertheless it was not possible to distinguish between true saturation of the initial rate of uptake and loss of linearity in uptake with time due to blebbing of hepatocytes; blebbing was previously shown to be both time and dose dependent (RUNNEGAR et al., 1981 ; RUNNEGAR and FALOONER, 1986). There was no significant difference (P greater than 0.05 by Student's t-test) in the initial rate of uptake when non-radioactive iodinated microcystin (300 and 600 nM) was

46

M . T . C. RLJNNEGAR et at . 20

0

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Microcystin concentration nM

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FIG . 2 . INITIAL RATE OF UPTAKE OF [~uI]-MICROCYSTIN HY HEPATOCYTES AS A FUNCTION OF MICROCYSr1N CONCENTRATION. Cells (I x l06/ml) were incubated at 30°C with varying concentrations of microcystin . Samples

(0.5 ml) were taken every I S sec. Cell associated microcystin was measured as described in the legend for Fig. 1 and in Materials and Methods . The initial rate of uptake v; was calculated for the interval 30-60 sec . Results are expressed as mean±S .E.M . (n = 4). In some cases the S.E .M . values are smaller than the symbols .

substituted for the same concentration of native toxin in the standard assay . Microscopic examination of incubations with the mono or düodinated microcystin (RuxxEGAR et al., 1986) showed blebbed hepatocytes that could not be distinguished from cells blebbed by equivalent concentrations of native microcystin . The uptake was strongly dependent on temperature (Fig. 3). At 0°C the uptake was too small to be measured accurately, the apparent activation energy from the initial uptake velocity (v;) values obtained from Fig . 3 was 18.5 kcal/mole (77 kJ/mole). ( tZSIJ-microcystin uptake: the effect of organic anions and other compounds Addition of about 50 p,M bromsulphthalein (BSP), sodium deoxycholate, cholate, rifampicin, antanamide, iopodate, and iodipamide to suspensions of hepatocytes

0

1

2

3

Time min

4

5

6

FIG. 3 . TEMPERATURE UEPENUENCE OF THE [~~ SI]-MICROCYSTIN UPTAKE BY HEPATOCYTFS . Cells (1 x 10"/ml) were incubated with 19nM microcystin at 0°, 25°, 30°, 37°C . Cell associated microcystin was measured as described in the legend for Fig . 1 and in Materials and Methods . Results are expressed as mean f S .E.M . (n = 3) .

Microcystin Uptake by Hepatocytes TABLE

I.

EFFECT'

OF

INFIIBrrORS ON HEPATOCYTES

MICROCYSI'IN

47 UPTAKE

BY

Inhibition of initial rate of uptake* Inhibitor concentration (~M) 10 50 L0 (a) With 19 nM microcystin BSP Sodium deoxycholate (b) With 519 nM microcystin BSP Sodium deoxycholate

49 ±6 60 f4 76 tl 33 f4

15 tl 32 ±3 22 t3 l9 t6

8 ±2 15 fl 7~4 t0~2 103 f0~4

"Initial rate of uptake v; of microcystin by hepatocytes in the presence of inhibitors expressed as % of uninhibited controls. This percentage was calculated as % of the mean for the uninhbited uptake as measured for each cell preparation . Results are expressed as means f S.E .M (n = 3). 1 x 106 cells/ml were incubated at 30°C with non-toxic (19 nM), and toxic (519 nM) concentrations of microcystin. Inhibitors were added at the same time as microcystin. Uptake was measured by a quick centrifugation method as described in Materials and Methods.

(1 x 106 cells/ml) inhibited the initial rate of uptake and cumulative uptake of ['zsI]microcystin . For the first two of these compounds the concentration dependence of the inhibition of microcystin uptake was investigated (Table 1). The inhibition of v; (expressed as % of v; in the absence of inhibitor) was dependent on the concentration of the inhibitor. As well as inhibiting the uptake of microcystin, BSP at 50 ~M also prevented blebbing of hepatocytes, (Fig. 4) by toxic concentrations (500 nM) of microcystin. The same protection from blebbing by microcystin was seen by scanning electron microscopy when SO ~M sodium cholate, deoxycholate or rifampicin replaced BSP under the same experimental conditions. The increase in phosphorylase a activity that follows addition of microcystin was also inhibited by preincubation with these compounds (Fig. 5); the protection was seen up to 30 min after addition . The inhibitors decreased the total amount of toxin taken up by the cells when compared to incubations with toxin only . As an example, 50 ~M BSP decreased the total uptake of microcystin in 30 min from 72 pmoles/106 cells to 10 pmoles/ 106 cells when hepatocytes were incubated with 519 nM microcystin. Replacement of sodium chloride in the incubation buffer with choline chloride caused 30% decrease in the initial rate of uptake of 17 nM microcystin, while replacement with potassium chloride or sucrose had no significant effect on v;. DISCUSSION

Administration of the heptapeptide microcystin radiolabeled with ['zSIJ to rats and mice showed it to be rapidly cleared from the circulation and to be preferentially accumulated in the liver (RUNNEGAR er al., 1986 ; FALCONER et al., 1986). Similar accumulations of

48

M. T. C. RUNNEGAR et al.

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Microcystin Uptake by Hepatocytes

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Control 7 min

Toxin 7 min

Control TI min

Toxin T! min

FIG. S . MICROCYSI'IN AND PH08PHORYLA3E a AC'T'IVITY OF HEPATOCYTFS. Inhibition of phosphorylase a activation by microcystin (500 nM) in hepatocytes by 50 pM BSP, sodium cholate, deoxychoLTte, rifampicin . SO pM of the inhibitor was added to hepatocytes (2 .75 x 10° cells/ml) at 37°C 6 min before addition of 500 nM toxin . Control incubations contained the appropriate inhibitor only. Samples were taken at 7 min and at 27 min after the addition of microcystin for the measurement of phosphorylase a activity as in RuNNeGAR et at. (1987) . Results are expressed as mean f S.E.M . (n = 3) . For both time points, 7 min and 27 min, there was no significant difference by Student's t-test between incubations containing inhibitor only (no toxin controls), and incubations that included 500 nM microcystin (toxin). The activity of phosphorylase a was significantly increased ('P < 0.05) at 7 min and 27 min for 500 nM microcystin incubations containing no inhibitors when compared with buffer only controls .

microcystin in the liver have also been shown by BROOKS and Conn (1987) with [iaC]microcystin, and by Rosnvsox et al. (1989) with ~H]-microcystin. The present study demonstrates that the uptake of microcystin by freshly isolated rat hepatocytes occurs readily. Uptake of organic anions such as microcystin can occur by diffusion or by carrier mediated transport (PETZINGER et al., 1983), although the nature and specificity of the transport systems are, as yet, only partially characterized (FRtMMER and ZIEGLER, 1988). If the uptake of microcystin is by carrier mediated transport rather than by simple diffusion, it should be saturable. In this study this could not be shown unequivocally. Published values for the kinetic constants for carrier mediated uptake of bile acids, and organic anions into isolated hepatocytes vary somewhat in different studies, but in all cases the Km values reported for initial rates of uptake are 10 ~M or slightly more (STREMMEL and BERG, 1986; IGA and Kt.AASSErr, 1982; PETZnvGER et al ., 1983). If microcystin is transported in a similar way, then the expected Km for its initial rate of uptake would also be in the micromolar range. Unfortunately, micromolar concentrations of microcystin are about 100-fold greater than those sufficient to damage hepatocytes. Unlike the initial rate of uptake (v;) of microcystin at nanomolar concentrations which increased linearly with extracellular concentration, initial uptake at micromolar concentrations was lower. If this decreased uptake is interpreted as saturation rather than reflecting changes in cell membrane due to blebbing a ~ of 10 ~M and a V,~ of 100 pmoles/min/ 106 cells at 30°C is obtained. These Km and V,gx values are of the same order of magnitude as reported in the literature (STRE~L and BERG, 1986; IGA and KLAASSEN, 1982 ; PETZINGER et al., 1983). Both the initial and cumulative uptake of microcystin are temperature dependent with an apparent activation energy of l8 kcal/mole (77 kJ/mole) ; this value is consistent with

50

M . T . C. RUNNEGAR et at.

values expected for carrier mediated transport, and much higher than values usually obtained for simple diffusion. The strongest indication that uptake of microcystin is, at least in part, by carrier mediated transport comes from the inhibition studies. Compounds that protect hepatocytes from the toxic effects of microcystin (RUNNEGAR et al., 1981 ; Figs 4 and 5), also inhibit, at similar concentrations, its uptake by the cells. These compounds are either bile acids, substrates for the multispecific transporter of hepatocytes or inhibitors of the carrier mediated transport of bile acids (FRIMMER et al., 1980 ; PETZINGER et al., 1983 ; FRIMMER and ZIEGLER, 1988). If the uptake of microcystin was by simple diffusion, it is not possible to amve at a mechanism to explain the inhibition of uptake by all these compounds. In contrast, this inhibition can be easily explained if microcystin was also, at least in part, taken up by one of the transporters of organic anions present in hepatocytes. Consistent with this conclusion is the earlier observation that blebbing of hepatocytes by higher microcystin doses (1-101cM) was at best only partially prevented by BSP, rifampicin and sodium deoxycholate (RUNNEGAR et al., 1981). Microcystin radiolabeled with 'ZSI and the equivalent non-radioactive iodinated derivatives had previously been shown to retain the same biological activity as the native toxin in vivo (RUNNEGAR et al., 1986). Here it was shown that the uptake of the iodinated derivatives by hepatocytes cannot be distinguished from that of the native toxin. In addition, similar blebbing of cells results from toxic doses of native or iodinated microcystins. 'ZSI-labeled microcystin is therefore a useful tool, both in vivo and in vitro, for future studies of the interaction, metabolism and fate of the toxin at the molecular level . Hepatocytes can concentrate toxin with respect to the extracllular medium . With morphologically normal hepatocytes the intracellular microcystin concentration increased with time to 0.90 ~M at 60 min (from Fig. 1A, using a cell volume of 6.2 ~1/ 106 cells (Aw et al., 1986)), a greater than 80-fold concentration factor with respect to the medium . Even when hepatocytes are blebbed there is accumulation of toxin. For example, in Fig. 1 B the cells contain 4.7 ~M microcystin, a 12-fold higher concentration with respect to the medium. The findings reported here support the conclusion that microcystin uptake by freshly isolated rat hepatocytes is carrier mediated, explaining the organ specificity of the toxin in vivo. Further studies are needed to identify which particular system of the many characterized hepatic transport mechanisms is involved in microcystin uptake . Acknowtedgement.~This work was supported in part by a grant from the Australian Research Grants Scheme . We thank ToM BucRLEV and Mike F~ttxwoRrx for excellent technical assistance .

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51

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