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Rhodamine exclusion activity in primary cultured turbot (Scophthalmus maximus) hepatocytes
Marine Environmental Research 54 (2002) 443–447 www.elsevier.com/locate/marenvrev
Rhodamine exclusion activity in primary cultured turbot (Scophthalmus maximus) hepatocytes Renaud Tutundjian*, Christophe Minier, Frank Le Foll, Franc¸ois Leboulenger Laboratoire d’Ecotoxicologie, UPRES-EA 3222, Universite´ du Havre, BP 540, 76058 Le Havre, France
Abstract Cellular detoxification by direct processes has been investigated in fish by studying the ability of hepatocytes prepared from juvenile aquarium-reared turbot (Scophthalmus maximus) to actively exclude the fluorescent dye rhodamine B (RB). Cell viability was studied by measurements of non-specific esterase activity using fluorescein diacetate. This revealed that turbot hepatocytes can be cultured for a few days with a viability decreasing to 38% after 24 h. The 24-h cultured cells have been used to study the rhodamine B exclusion activity using confocal laser microscopy. Hepatocytes accumulated the dye in a competitive manner with verapamil, thus suggesting that they express a transport system similar to the P-glycoproteinmediated multixenobiotic resistance process. Incubation of cells with 1 mM RB and 20 mM verapamil led to a 26% increase of cellular fluorescence as compared to the accumulation in absence of competitor. Rhodamine B accumulated in the whole cytoplasm, with more concentrated areas that might correspond to the lysosomal compartment and the cell membrane. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Turbot; Scophthalmus maximus; P-glycoprotein; MXR; Lysosomes
Multidrug resistance (MDR) or multxenobiotic resistance (MXR) is one of the most intriguing phenomenon in cell biology. Indeed, this mechanism is responsible for simultaneous cross-resistance against a variety of structurally and functionally unrelated toxic compounds (Gottesman & Pastan, 1993). If the mechanism of action is known to involve the expression of high molecular weight membrane proteins (P-glycoproteins) which actively transport the xenobiotics out of the cell (thus leading
* Corresponding author. Tel. :+33-232-744303 ; fax : +33-232-744314. E-mail address: [email protected] (R. Tutundjian). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00161-7
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R. Tutundjian et al. / Marine Environmental Research 54 (2002) 443–447
to a decrease in toxicity) (Gros, Neriah, Croop, & Housman, 1986), the underlying mechanism which allows a single protein to handle such a variety of compounds is still a matter of debate and leads to numerous hypothesis (Eytan & Kuchel, 1999). The P-glycoprotein-driven resistance mechanism is spread over all the cellularbased life. Its corresponding mdr genes have been highly conserved during evolution and their expression could correspond to a first line of defence which, in addition, might be indicative of the exposure to some xenobiotics (Epel, 1998). In fish, DNA fragments coding for MXR-like proteins have been sequenced in the winter flounder, the European flounder and the killifish (reviewed in Bard, 2000). Recently, we found that turbot hepatocytes express a MXR-related gene which partial product (144 amino-acids including an ATP-binding site) shares about 80% homology with the mammalian mdr class I proteins (EMBL AC: AJ291813). Here we report the first examination and measurements of rhodamine B accumulation in primary cultured turbot hepatocytes. Indeed, rhodamine is a well-known MDR/ MXR substrate and its accumulation can be modulated by specific inhibitors such as verapamil, thus giving a good indication of the MDR phenotype (Gottesman & Pastan, 1993). Livers were dissected from juvenile turbot (8–12 cm long) and chopped with a razor blade. Hepatocytes were isolated using collagenase (0.25 mg ml 1), for 20 min at 20 C and filtered through a 150-mm mesh filter. Cells were then incubated in DMEM/F12 supplemented with 1% antibiotics, 5 mM glutamine, 0.2 mg ml 1 albumin, 1% of the cell culture complement G5 (Life Technologies) and 2% of the Ultroser steroid-free (USF) serum (Biosepra) at 18 C on a rotating platform (60 rpm). Initial cell viability was evaluated both by trypan blue exclusion and nonspecific esterase activity, using fluorescein-diacetate (FDA) as substrate as described in Larsson and Nygren (1989). Fluorescence apparition was assessed using a microplate reader with settings as follow: excitation filter, 485 nm, emission filter, 530 nm. After 24 h, the hepatocytes were plated on polylysine coated slips for at least 1 h. Prior to measurements, cells were incubated for 30 min with or without 20 mM verapamil and then for another 30 min with 1 mM rhodamine B ( 20 mM verapamil). Images of the live cells that remained attached were finally taken with a laserscan confocal microscope (laser excitation 568 nm, barrier filter emission 590 nm) and single cell fluorescence was measured using a computerised image analysis system (Silicon Graphics). Results showed that FDA activity is closely related to the cell concentration for up to 100,000 hepatocytes/well (R2=0.957, P < 0.001, Fig. 1a). According to these results and because of the linear relationship between FDA and other methods applied for assessing cell viability (Larsson & Nygren, 1989), this rapid and sensitive method was further used to study the survival rate of the hepatocytes over time. Esterase activity fell to 38% after the first day of incubation (Fig. 1b) and then slightly decreased to 25% after 48 h. This cell survival rate, although rather small, is frequently observed in primary cultured hepatocytes (Guillouzo, 1995). Better results often involve in situ perfusion of liver with collagenase but it was not possible to apply this protocol to juvenile fish. FDA activity was also used to assess the potential toxicity of verapamil during the incubation procedure and results showed
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that it had no effect on the hepatocytes over a 1-h exposure time even at concentrations up to 40 mM verapamil (data not shown). Confocal microscopy allowed to visualise the RB accumulation in hepatocytes. Different patterns of dye accumulation could be observed depending on the presence or absence of verapamil in the incubation medium (Fig. 2). Fluorescence was significantly enhanced (+ 26% as compared to control; P < 0.001, non-paired Student t-test, n=20) when the MXR inhibitor was present (Fig. 2). This increase in dye accumulation is similar to that observed in Mytilus edulis hemocytes (Minier &
Fig. 1. Non-specific esterase activity of turbot hepatocytes assessed using 20 mM fluorescein-diacetate (FDA) as substrate. FDA hydrolysis led to a fluorescence intensity proportional to the concentration of live cells for up to 100,000 cells/well (a). Evolution of FDA activity during cell culture (b). Bars show confidence intervals (=0.05; n=4).
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R. Tutundjian et al. / Marine Environmental Research 54 (2002) 443–447
Moore, 1996) and might be the result of a competition mechanism between verapamil and rhodamine B. Rhodamine accumulated in the whole cytoplasm with an increased labelling in parts of the cellular membrane and some intracellular structures. The labelled membrane area may correspond to the secretory surface of the cell where MDR proteins are known to be preferentially localised in mammalian cells (Croop et al., 1989) and could thus be due to the interaction of the dye with the MXR proteins. The intracellular spots might correspond to lysosomes which are the only labelled structures when exposing mussel hemocytes to rhodamine B (Minier & Moore, 1996). In this respect, the lysosomal accumulation could correspond to a secondary defence mechanism against compounds that overwhelm the MXR system (Moore & Wilows, 1998). Together, these results show that turbot hepatocytes are expressing an MXR system that can lower the intracellular rhodamine accumulation and suggest that this mechanism can be part of a defence mechanism against other substrates to be
Fig. 2. Rhodamine B accumulation in turbot hepatocytes incubated for 1 h without (a and b) or with (c) 20 mM verapamil, and with 1 mM rhodamine during the last 30 min of the incubation time. Incubation with verapamil led to a 26% increase of the cell fluorescence (d,confidence intervals, =0.05; n=20).
R. Tutundjian et al. / Marine Environmental Research 54 (2002) 443–447
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identified. The isolated cells are thus an interesting tool to assess the impact of individual or mixed compounds on the activity of the MXR system.
Acknowledgements This work is part of the European ‘‘Biological Evaluation of Environmental Pollution’’ (BEEP) program. Mr. R. Tutundjian is recipient of a research grant from the Re´gion Haute-Normandie. The authors are grateful to Dr. H. Vaudry and Mrs. C. Rousselle (INSERM U413, IFRMP 23, Rouen, France) for their help and advises on confocal microscopy. They also would like to thank Mr. M. Bonnet for his kind help during cell culture experiments.
References Bard, S. M. (2000). Aquatic Toxicology, 48, 357–389. Croop, J. M., Raymond, M., Haber, D., Devault, A., Arceci, R. J., Gros, P., & Housman, D. E. (1989). Molecular and Cellular Biology, 9, 1346–1350. Epel, D. (1998). Comparative Biochemistry and Physiology, A120, 23–28. Eytan, G., & Kuchel, P. W. (1999). International Review of Cytology, 190, 175–250. Gottesman, M. M., & Pastan, I. (1993). Annual Review of Biochemistry, 62, 385–427. Gros, P., Neriah, Y. B., Croop, J. M., & Housman, D. E. (1986). Nature, 323, 728–731. Guillouzo, A. In M. Adolphe, et al. (Eds.), Toxicologie cellulaire in vitro, me´thodes et applications (pp. 69– 120). Paris: Editions INSERM. Larsson, R., & Nygren, P. (1989). Anticancer Research, 9, 1111–1120. Minier, C., & Moore, M. N. (1996). Marine Ecology Progress Series, 142, 165–173. Moore, M. N., & Willows, R. I. (1998). Marine Environmental Research, 46, 509–514.
Rhodamine exclusion activity in primary cultured turbot (Scophthalmus maximus) hepatocytes Renaud Tutundjian*, Christophe Minier, Frank Le Foll, Franc¸ois Leboulenger Laboratoire d’Ecotoxicologie, UPRES-EA 3222, Universite´ du Havre, BP 540, 76058 Le Havre, France
Abstract Cellular detoxification by direct processes has been investigated in fish by studying the ability of hepatocytes prepared from juvenile aquarium-reared turbot (Scophthalmus maximus) to actively exclude the fluorescent dye rhodamine B (RB). Cell viability was studied by measurements of non-specific esterase activity using fluorescein diacetate. This revealed that turbot hepatocytes can be cultured for a few days with a viability decreasing to 38% after 24 h. The 24-h cultured cells have been used to study the rhodamine B exclusion activity using confocal laser microscopy. Hepatocytes accumulated the dye in a competitive manner with verapamil, thus suggesting that they express a transport system similar to the P-glycoproteinmediated multixenobiotic resistance process. Incubation of cells with 1 mM RB and 20 mM verapamil led to a 26% increase of cellular fluorescence as compared to the accumulation in absence of competitor. Rhodamine B accumulated in the whole cytoplasm, with more concentrated areas that might correspond to the lysosomal compartment and the cell membrane. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Turbot; Scophthalmus maximus; P-glycoprotein; MXR; Lysosomes
Multidrug resistance (MDR) or multxenobiotic resistance (MXR) is one of the most intriguing phenomenon in cell biology. Indeed, this mechanism is responsible for simultaneous cross-resistance against a variety of structurally and functionally unrelated toxic compounds (Gottesman & Pastan, 1993). If the mechanism of action is known to involve the expression of high molecular weight membrane proteins (P-glycoproteins) which actively transport the xenobiotics out of the cell (thus leading
* Corresponding author. Tel. :+33-232-744303 ; fax : +33-232-744314. E-mail address: [email protected] (R. Tutundjian). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00161-7
444
R. Tutundjian et al. / Marine Environmental Research 54 (2002) 443–447
to a decrease in toxicity) (Gros, Neriah, Croop, & Housman, 1986), the underlying mechanism which allows a single protein to handle such a variety of compounds is still a matter of debate and leads to numerous hypothesis (Eytan & Kuchel, 1999). The P-glycoprotein-driven resistance mechanism is spread over all the cellularbased life. Its corresponding mdr genes have been highly conserved during evolution and their expression could correspond to a first line of defence which, in addition, might be indicative of the exposure to some xenobiotics (Epel, 1998). In fish, DNA fragments coding for MXR-like proteins have been sequenced in the winter flounder, the European flounder and the killifish (reviewed in Bard, 2000). Recently, we found that turbot hepatocytes express a MXR-related gene which partial product (144 amino-acids including an ATP-binding site) shares about 80% homology with the mammalian mdr class I proteins (EMBL AC: AJ291813). Here we report the first examination and measurements of rhodamine B accumulation in primary cultured turbot hepatocytes. Indeed, rhodamine is a well-known MDR/ MXR substrate and its accumulation can be modulated by specific inhibitors such as verapamil, thus giving a good indication of the MDR phenotype (Gottesman & Pastan, 1993). Livers were dissected from juvenile turbot (8–12 cm long) and chopped with a razor blade. Hepatocytes were isolated using collagenase (0.25 mg ml 1), for 20 min at 20 C and filtered through a 150-mm mesh filter. Cells were then incubated in DMEM/F12 supplemented with 1% antibiotics, 5 mM glutamine, 0.2 mg ml 1 albumin, 1% of the cell culture complement G5 (Life Technologies) and 2% of the Ultroser steroid-free (USF) serum (Biosepra) at 18 C on a rotating platform (60 rpm). Initial cell viability was evaluated both by trypan blue exclusion and nonspecific esterase activity, using fluorescein-diacetate (FDA) as substrate as described in Larsson and Nygren (1989). Fluorescence apparition was assessed using a microplate reader with settings as follow: excitation filter, 485 nm, emission filter, 530 nm. After 24 h, the hepatocytes were plated on polylysine coated slips for at least 1 h. Prior to measurements, cells were incubated for 30 min with or without 20 mM verapamil and then for another 30 min with 1 mM rhodamine B ( 20 mM verapamil). Images of the live cells that remained attached were finally taken with a laserscan confocal microscope (laser excitation 568 nm, barrier filter emission 590 nm) and single cell fluorescence was measured using a computerised image analysis system (Silicon Graphics). Results showed that FDA activity is closely related to the cell concentration for up to 100,000 hepatocytes/well (R2=0.957, P < 0.001, Fig. 1a). According to these results and because of the linear relationship between FDA and other methods applied for assessing cell viability (Larsson & Nygren, 1989), this rapid and sensitive method was further used to study the survival rate of the hepatocytes over time. Esterase activity fell to 38% after the first day of incubation (Fig. 1b) and then slightly decreased to 25% after 48 h. This cell survival rate, although rather small, is frequently observed in primary cultured hepatocytes (Guillouzo, 1995). Better results often involve in situ perfusion of liver with collagenase but it was not possible to apply this protocol to juvenile fish. FDA activity was also used to assess the potential toxicity of verapamil during the incubation procedure and results showed
R. Tutundjian et al. / Marine Environmental Research 54 (2002) 443–447
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that it had no effect on the hepatocytes over a 1-h exposure time even at concentrations up to 40 mM verapamil (data not shown). Confocal microscopy allowed to visualise the RB accumulation in hepatocytes. Different patterns of dye accumulation could be observed depending on the presence or absence of verapamil in the incubation medium (Fig. 2). Fluorescence was significantly enhanced (+ 26% as compared to control; P < 0.001, non-paired Student t-test, n=20) when the MXR inhibitor was present (Fig. 2). This increase in dye accumulation is similar to that observed in Mytilus edulis hemocytes (Minier &
Fig. 1. Non-specific esterase activity of turbot hepatocytes assessed using 20 mM fluorescein-diacetate (FDA) as substrate. FDA hydrolysis led to a fluorescence intensity proportional to the concentration of live cells for up to 100,000 cells/well (a). Evolution of FDA activity during cell culture (b). Bars show confidence intervals (=0.05; n=4).
446
R. Tutundjian et al. / Marine Environmental Research 54 (2002) 443–447
Moore, 1996) and might be the result of a competition mechanism between verapamil and rhodamine B. Rhodamine accumulated in the whole cytoplasm with an increased labelling in parts of the cellular membrane and some intracellular structures. The labelled membrane area may correspond to the secretory surface of the cell where MDR proteins are known to be preferentially localised in mammalian cells (Croop et al., 1989) and could thus be due to the interaction of the dye with the MXR proteins. The intracellular spots might correspond to lysosomes which are the only labelled structures when exposing mussel hemocytes to rhodamine B (Minier & Moore, 1996). In this respect, the lysosomal accumulation could correspond to a secondary defence mechanism against compounds that overwhelm the MXR system (Moore & Wilows, 1998). Together, these results show that turbot hepatocytes are expressing an MXR system that can lower the intracellular rhodamine accumulation and suggest that this mechanism can be part of a defence mechanism against other substrates to be
Fig. 2. Rhodamine B accumulation in turbot hepatocytes incubated for 1 h without (a and b) or with (c) 20 mM verapamil, and with 1 mM rhodamine during the last 30 min of the incubation time. Incubation with verapamil led to a 26% increase of the cell fluorescence (d,confidence intervals, =0.05; n=20).
R. Tutundjian et al. / Marine Environmental Research 54 (2002) 443–447
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identified. The isolated cells are thus an interesting tool to assess the impact of individual or mixed compounds on the activity of the MXR system.
Acknowledgements This work is part of the European ‘‘Biological Evaluation of Environmental Pollution’’ (BEEP) program. Mr. R. Tutundjian is recipient of a research grant from the Re´gion Haute-Normandie. The authors are grateful to Dr. H. Vaudry and Mrs. C. Rousselle (INSERM U413, IFRMP 23, Rouen, France) for their help and advises on confocal microscopy. They also would like to thank Mr. M. Bonnet for his kind help during cell culture experiments.
References Bard, S. M. (2000). Aquatic Toxicology, 48, 357–389. Croop, J. M., Raymond, M., Haber, D., Devault, A., Arceci, R. J., Gros, P., & Housman, D. E. (1989). Molecular and Cellular Biology, 9, 1346–1350. Epel, D. (1998). Comparative Biochemistry and Physiology, A120, 23–28. Eytan, G., & Kuchel, P. W. (1999). International Review of Cytology, 190, 175–250. Gottesman, M. M., & Pastan, I. (1993). Annual Review of Biochemistry, 62, 385–427. Gros, P., Neriah, Y. B., Croop, J. M., & Housman, D. E. (1986). Nature, 323, 728–731. Guillouzo, A. In M. Adolphe, et al. (Eds.), Toxicologie cellulaire in vitro, me´thodes et applications (pp. 69– 120). Paris: Editions INSERM. Larsson, R., & Nygren, P. (1989). Anticancer Research, 9, 1111–1120. Minier, C., & Moore, M. N. (1996). Marine Ecology Progress Series, 142, 165–173. Moore, M. N., & Willows, R. I. (1998). Marine Environmental Research, 46, 509–514.