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Down-regulation of fibronectin in rainbow trout gonadal cells exposed to retinoic acid
Aquatic Toxicology 48 (2000) 119 – 125 www.elsevier.com/locate/aquatox
Down-regulation of fibronectin in rainbow trout gonadal cells exposed to retinoic acid M.G. Miller a, C.M. Kapron a, C.D. Metcalfe b,*, L.E.J. Lee c,1 a Department of Biology, Trent Uni6ersity, Peterborough, Ont., Canada K9J 7B8 En6ironmental and Resource Studies Program, Trent Uni6ersity, Peterborough, Ont., Canada K9J 7B8 c Department of Veterinary Anatomy, Western College of Veterinary Medicine, Uni6ersity of Saskatchewan, Saskatoon, Sask., Canada S7N 5B4 b
Received 19 February 1999; received in revised form 3 June 1999; accepted 11 June 1999
Abstract Exposure of fish to some environmental contaminants results in alterations to the levels of retinoid (Vitamin A) stores, which could result in an increase in cellular concentrations of biologically active metabolites such as retinoic acid (RA). However, a link has not been established between changes in retinoid metabolism and impacts on the health of biota. In vitro studies with mammalian cells have demonstrated a relationship between exposure to RA and expression of the extracellular matrix protein, fibronectin (FN); a protein critical for cell migration, adhesion, and transformation. In this study, in vitro exposures of rainbow trout gonadal cells (RTG-2) to RA reduced levels of FN in culture medium; as measured using SDS-PAGE and immunoblot analysis with antisera prepared against RTG-2 cellular fibronectin. This apparent down-regulation of FN secretion occurred in a dose-dependent manner over a range of RA concentrations (10 − 10 –10 − 6 M). FN down-regulation was not accompanied by changes in the morphology of RTG-2. Future studies should be directed at determining the relationships between retinoid metabolism and FN expression and the potential effects of contaminant-induced changes to vitamin A metabolism on the health of fish. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Fibronectin; Rainbow trout; Retinoic acid
1. Introduction Fish from contaminated aquatic environments are often in poor health, as indicated by reduced growth, reproductive failure, immunosuppression, * Corresponding author. Tel.: 1-705-748-1272; fax: + 1-705748-1569. 1 Current address: Department of Biology, Wilfrid Laurier University, Waterloo, Ont., Canada N2L 3C5.
skin lesions, deformities, altered hormone levels, changes to the activity of metabolic enzymes and altered concentrations of vitamins (Munkittrick and Dixon, 1989; Arkoosh et al., 1991; Munkittrick et al., 1992; Branchaud et al., 1995; Foster, 1995; Arcand-Hoy and Metcalfe, 1999). Several investigators have observed alterations to the homeostasis of Vitamin A compounds (i.e. retinoids) in fish from areas contaminated with
0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 9 9 ) 0 0 0 5 2 - 1
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persistent organic contaminants. Branchaud et al. (1995) observed reduced levels of hepatic retinol and retinyl palmitate in white suckers (Catostomus commersoni ) sampled near urban areas of the St. Lawrence River. Malformations and eye deformities were more prevalent in sucker populations from contaminated sites. In a recent study of brown bullhead (Ameiurus nebulosus) from various sites in the lower Great Lakes, Arcand-Hoy and Metcalfe (1999) observed reduced concentrations of hepatic retinyl palmitate in bullheads from contaminated sites. Bullheads from contaminated areas had high prevalences of skin lesions. These field data are consistent with a hypothesis that retinoid homeostasis is altered in fish exposed to environmental contaminants. Several laboratory studies have examined the impacts of halogenated aromatic hydrocarbons (HAHs) such as polyhalogenated dioxins and biphenyls on vitamin A homeostasis in rodents (Jensen and Zile, 1988; Brouwer et al., 1989a; Hakansson et al., 1994). In studies with seals (Phoca 6itulina) fed PCB-contaminated fish from the Wadden Sea, Brouwer et al. (1989b) detected reduced plasma retinol levels. Palace and Brown (1994) exposed lake trout to 3,3%,4,4%,5-pentachlorobiphenyl and observed reduced concentrations of retinol, dehydroretinol and retinyl palmitate in the liver and elevated retinyl palmitate in the kidney. These studies support the hypothesis that exposure to persistent organic contaminants, and in particular HAHs, results in a decrease in retinoid stores and an increase in excretion of vitamin A catabolites (Jensen and Zile, 1988). Retinoids are essential to a variety of physiological processes, including growth, maintenance of epithelial tissues and vision, and regulation of embryogenesis and spermatogenesis (Zile, 1992). Alterations in retinoid homeostasis can induce many effects; including growth retardation, teratogenesis, epidermal lesions, ocular changes and sterility (Bhagavan, 1992). Similarities between these toxic symptoms and some of the health effects observed in fish from contaminated environments are evident; supporting the hypothesis that some of these health effects are induced through mechanisms that involve alterations to Vitamin A homeostasis.
Although exposure to HAHs appears to alter retinoid metabolism, there is no clear mechanistic link between retinoid modulation and specific toxic effects in organisms. Recent in vitro studies have shown that retinoid metabolites can induce changes in the cellular production of fibronection (FN) in mammalian cells (Scita and Wolf, 1994; Scita et al., 1996); suggesting a link between retinoid homeostasis and maintenance of this extracellular matrix protein. Through various molecular binding interactions, FN is involved in a range of cellular processes; including cell adhesion, migration, transformation and differentiation (Mosher, 1984). Thus, alterations to FN levels could have profound effects upon embryonic development, cellular differentiation and maintenance of tissues. For instance, immunohistochemical analysis has shown that FN deposition is increased along the pathways of neural crest cell migration in fish embryos; leading to speculation that FN may be involved in both the initiation and guidance of neural crest cells during their migration (Sadaghiani et al., 1994). FN is also involved in the wound healing process, as it is a component of the initial fibrin matrix deposited during blood clotting (Mosher, 1984). Several in vitro studies with mammalian and avian cells have linked exposure of cells to retinoic acid (RA) with changes in the production of cellular FN (Zerlauth and Wolf, 1984; Kim and Wolf, 1987; Horton et al., 1987; Scheidl et al., 1991; Scita and Wolf, 1994; Scita et al., 1996). Although transcription of the gene for FN is regulated by binding of RA to RA responsive elements (Wolf, 1990), these in vitro studies have shown that the direction of regulation appears to be dependent on the cell type studied. Work by Shanker and Sawhney (1996) with primary cultures of bovine lens epithelial cells showed that RA up-regulated FN by increasing transcription. Horton et al. (1987) observed that FN was upregulated in chick chondrocytes by treatment with RA, and that up-regulation was accompanied by flattening and elongation of chondrocytes so that they resembled fibroblastic cells. In all in vitro studies with mammalian fibroblasts, FN was down-regulated in a dose-dependent manner by exposure to exogenous RA (Zerlauth and Wolf, 1984; Scheidl et al., 1991; Scita and Wolf, 1994).
M.G. Miller et al. / Aquatic Toxicology 48 (2000) 119–125
In this study, in vitro assays were conducted to determine if RA can alter FN production in fish cells. The rainbow trout gonadal cell line (RTG-2) was chosen as an in vitro model because it is fibroblastic in morphology and produces large quantities of FN (Lee and Bols, 1991). RTG-2 cultures were exposed to varying concentrations of RA in serum-free medium and the medium was analyzed for FN levels using SDS-PAGE electrophoresis, followed by immunoblot analysis with antibodies raised against RTG-2 fibronectin to confirm the identity of the putative FN band. Based on studies with mammalian fibroblasts (Scita and Wolf, 1994; Scita et al., 1996), it was hypothesized that RA would down-regulate FN in a dose-dependent manner. This study is the first step in determining whether there may be a link between contaminant-induced alterations to retinoid homeostasis and health effects in fish that may be related to alterations to the extracellular matrix.
2. Methods
2.1. Cell culture and exposure to RA RTG-2 cells were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in 25 or 75 cm2 polystyrene flasks in Leibovitz’s L-15 medium (Gibco-BRL) with 10% fetal bovine serum (HyClone) and penicillin/streptomycin antibiotic mixture (100 U/ml penicillin: 100 mg/ml streptomycin). Cells were cultured in the dark at 22 9 2°C in an atmosphere of air, and were grown to confluency with fluid renewal each week. For RA exposures, 3× 105 cells were seeded into 75 cm2 flasks and then grown to confluency. Confluent monolayers were rinsed 3 times with serum-free L-15 medium to remove serum proteins, and 10 ml of serum-free medium was added that contained one of 10 − 5, 10 − 6, 10 − 7, 10 − 8, 10 − 9, and 10 − 10 M RA (Sigma) in dimethylsulfoxide (DMSO) or DMSO carrier (dissolved in media to 0.0001% v/v). Cells were maintained in this medium for 5 days prior to analysis of FN.
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2.2. SDS-PAGE analysis of FN The medium was collected from the cultures and centrifuged for 5 min with a benchtop clinical centrifuge to remove cellular debris. Solutions were then concentrated using Ultrafree-15 centrifugation filters (Millipore), which were centrifuged for 1 h at 2000× g at room temperature (22°C). The concentrated medium was pipetted out of the filter and either used immediately or stored at − 80°C for later analysis. Total protein concentration in the concentrated medium was determined using a Bio-Rad protein microassay. Samples were equalized for total protein and 10 ml subsamples were electrophoresed under reducing conditions (5% bmercaptoethanol) on 6% or 7.5% SDS-polyacrylamide gels, according to the method of Laemmli (1970). The relative mobilities of resolved proteins were compared to those of Kaleidoscope prestained molecular weight markers (BioRad). Following electrophoresis, gels were silver stained to visualize protein bands using the procedure of Blum et al. (1987); modified because of high protein levels by eliminating the final step of the staining procedure. Stained gels were scanned using a Canon DeskJet III Scanner, and scanned images were saved as .tif format files. The uncalibrated optical densities of bands were measured by analysis of.tif files using a shareware program, NIH-Image 1.56 (NIH, Bethesda, MD). Putative FN bands were identified by their position at 220 kDa relative to molecular weight markers. The relative densities of these bands were corrected for between-lane variation by dividing by the density of a reference band common to each lane and consistent in density across all lanes. Note that loadings on all lanes were equalized for protein content.
2.3. Immunodetection of FN To positively identify putative FN bands, samples were subjected to immunoblot analysis using a rabbit anti-trout FN polyclonal antibody. Antibody was prepared by purifying fibronectin from conditioned media of RTG-2 as described by Lee
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and Bols (1991). Briefly, the conditioned media of RTG-2 that had been grown in the absence of serum was collected and subjected to gelatin affinity chromatography. Column eluates were monitored for protein content and the putative fibronectin fraction was collected, dialyzed and tested for biological activity in terms of cell attachment (Lee and Bols, 1991). This preparation was then subjected to preparative SDS gel electrophoresis. A prominent doublet band was noted at 220 kDa which was eluted out of the gels using a BioRad electroeluter following manufacturer’s instructions. The eluted protein was dissolved in physiological saline and a schedule of injections were performed in rabbits following standard operating procedures for antisera production at the Animal Care Unit of the Western College of Veterinary Medicine, University of Saskatchewan. Anti-cellular fibronectin was obtained which recognized trout fibronectin as well as various commercially purchased mammalian fibronectins.
After SDS-PAGE of medium as described above, proteins were transferred in buffer (25 mM Tris; 192 mM glycine; 20% MeOH v/v) onto PVDF membranes (Immobilon-P, Millipore) on a Bio-Rad electroblot apparatus (100 V constant for 1 h). The membranes were blocked in a 7.5% purified non-fat milk solution (Bio-Rad) for 30 min at 37°C. Rabbit anti-trout FN antibodies at a dilution of 1:1000 (in 5% milk) were applied for 1 h at room temperature with agitation, followed by three 10 min washes in TBS-T (20 mM Tris, 137 mM NaCl, 1% v/v Tween-20, pH 7.6) under the same conditions. The blots were then incubated with horseradish-peroxidase labelled secondary antibodies (goat anti-rabbit IgG1, Jackson Immunoresearch Labs) at 1:5000 dilution in a 5% milk/TBS-T solution. The membranes were then washed (3× 10 min) in TBS-T and proteins were detected using the ECL Western Blotting Detection system (Amersham).
3. Results and discussion
Fig. 1. Polyacrylamide gels (silver stained) showing the putative fibronectin (FN) band prepared from samples of rainbow trout gonadal cells (RTG-2) exposed to retinoic acid (RA) at concentrations of 10 − 8, 10 − 7 and 10 − 6 M. Also indicated are the molecular weight (MW) markers at 71, 133 and 202 kDa and the reference band used in the semi-quantitative analysis of optical densities of the putative FN band.
Exposure of RTG-2 to RA resulted in a decrease in the density of the putative FN bands; especially at exposure concentrations \10 − 8 M (Fig. 1). As illustrated in Fig. 2, the decline in density of the bands was concentration dependent; fitting a regression line of y= − 0.050 ln x− 0.088 (r 2 = 0.92). Immunoblot analysis of culture media confirmed the tentative identification of the FN band in earlier experiments, and also showed a dose-dependent decrease in FN over a concentration range of 10 − 8 –10 − 6 M RA (Fig. 3). This response is consistent with down-regulation of FN secretion into culture media in response to increasing RA concentrations. RTG-2 in culture appeared fibroblastic in morphology; characterized by a flattened appearance with multiple protrusions of the cytoplasm. In culture, the cells grew from several foci on the dish in a spiral pattern. In treatments with RA, there were no differences in density or attachment of RTG-2, and there was no alteration to the morphology of the cells. In mammalian chondrocytes, FN up-regulation was accompanied by dis-
M.G. Miller et al. / Aquatic Toxicology 48 (2000) 119–125
Fig. 2. Relative optical density of putative fibronectin (FN) bands in polyacrylamide gels (silver stained) using samples prepared from rainbow trout gonadal cells (RTG-2) in control treatment and in treatments with retinoic acid (RA) at concentrations of between 10 − 9 and 10 − 5 M. The arrow on the y-axis indicates the optical density of the band from the control treatment.
Fig. 3. Representative immunoblot of polyacrylamide gels showing fibronectin (FN) bands identified by immunoblot analysis with rabbit anti-trout FN polyclonal antisera in samples prepared from rainbow trout gonadal cells (RTG-2) exposed to various concentrations (10 − 10 –10 − 8 M) of retinoic acid (RA).
tinct flattening of cells to a more fibroblastic form (Horton et al., 1987); consistent with the role of the extracellular matrix, and FN in particular, in regulating cell morphology (Hynes and Yamada, 1982). The lack of a change in morphology in RTG-2 in treatments where RA-induced downregulation of FN was observed may indicate that the residual extracellular matrix (ECM) proteins remaining from pre-treatment continued to support the normal morphology of the cells through-
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out the 5 day exposure period. Longer exposures to RA may induce changes in the morphology of RTG-2. While the kinetics of release of FN and ECM proteins is well known for other cell types (Scita et al., 1996), RTG-2 may not follow the same pattern as mammalian cell lines. These results with RTG-2 are consistent with the down-regulation of FN observed in in vitro studies with mammalian fibroblasts (Scita and Wolf, 1994; Scita et al., 1996), but are contrary to other studies that show RA-induced up-regulation of FN in chick chondrocytes (Horton et al., 1987) and bovine lens epithelial cells (Shanker and Sawhney, 1996). The reasons for the differences between cell types is not known, but may be associated with differences in FN gene expression following binding of RA with RA responsive elements (Shanker and Sawhney, 1996). It appears from this study that RTG-2 resemble mammalian fibroblasts in the pattern of regulation of FN expression. This study also illustrates the potential for using RTG-2 as an in vitro model for studies of the effects of retinoids on gonadal differentiation and development. It is difficult to interpret these in vitro results with RTG-2 in terms of potential in vivo responses in fish. Different tissues may vary in RA-induced regulation of FN expression. In addition, RA is rapidly metabolized in vivo because of the potent biological effects of this retinoid compound at various locations in the body (Blomhoff et al., 1991). For instance, as a morphogen during embryonic development, RA is transiently maintained at specific target tissues such as limb buds to control positional morphology (Giguere et al., 1987). Therefore, one needs to know more about the metabolism of retinoids in fish in order to determine if RA is mobilized in response to reduced levels of the storage forms of retinoids and whether RA is ‘delivered’ to tissues in concentrations sufficient to alter FN expression. While RA concentrations in human plasma are approximately 10 − 9 M (Bhagavan, 1992), the endogenous concentrations of RA in fish plasma and tissues have not been studied. Finally, FN occurs in several different forms that vary between species and even within a single organism; differing in structural features such as binding sites and
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glycosylation locations (Yamada, 1991). There may be differences in responses to RA that are related to structural differences in FN. Field studies have shown that retinoids are mobilized in fish exposed to environmental contaminants (Branchaud et al., 1995; Arcand-Hoy and Metcalfe, 1999), and this has been supported by experimental studies with fish exposed to PCB compounds (Palace and Brown, 1994). In birds and mammals exposed to HAHs, mobilization of retinoid stores in the liver occurs through metabolism of non-polar (i.e. fat-soluble) retinyl esters to more polar retinol (Spear et al., 1990; Zile, 1992). It is not clear whether higher levels of serum retinol can lead to altered concentrations of cellular RA. However, in vitro studies with rat liver microsomes showed that both retinol and retinal are metabolically converted into retinoic acid (Napoli and Race, 1990). Considerablely more work is required to determine whether there is a possible link between contaminant-induced mobilization of retinoid stores in organisms and changes to cellular RA levels that could have potent biological effects.
4. Conclusions In this study, RA appeared to down regulate FN in a dose-dependent manner in rainbow trout gonadal cells. This molecular response was not accompanied by changes in cell morphology under the in vitro conditions used in this study. More work is required to determine if these responses can be observed in vivo in fish species, and to determine whether RA-induced alterations in FN expression are linked to specific health effects in fish. Further study is also needed to characterize changes to vitamin A metabolism (including cellular production of RA) caused by exposure of fish and other biota to environmental contaminants.
Acknowledgements Katharine Haberstroh helped with laboratory procedures. This work was supported by Research
Grants from the Natural Sciences and Engineering Research Council to Chris Metcalfe and to Carolyn Kapron.
References Arcand-Hoy, L.D., Metcalfe, C.D., 1999. Biomarkers of exposure of brown bullheads (Ameirus nebulosus) to contaminants in the lower Great Lakes, North America. Environ. Toxicol. Chem. 18, 740 – 749. Arkoosh, M.R., Casillas, E., Clemons, E., McCain, B., Varanasi, U., 1991. Suppression of immunological memory in juvenile chinook salmon (Oncorhynchus tshawytscha) from and urban estuary. Fish Shellfish Immunol. 23, 112 – 123. Bhagavan, N.V., 1992. Medical Biochemistry. Jones and Bartlett, Boston, p. 145. Blomhoff, R., Green, M.H., Balmer Green, J., Berg, T., Norum, K.R., 1991. Vitamin A metabolism: new perspectives on absorption, transport and storage. Physiol. Rev. 71, 951 – 990. Blum, H., Beir, H., Gross, H., 1987. Improved silver-staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93 – 99. Branchaud, A., Gendron, A., Fortin, R., Anderson, P.D., Spear, P.A., 1995. Vitamin A stores, teratogenesis, and EROD activity in white sucker, Catostomus commersoni, from Riviere des Prairies near Montreal and a reference site. Can. J. Fish. Aquat. Sci. 52, 1703 – 1713. Brouwer, A., Hakansson, H., Kukler, A., Van Den Berg, K.J., Ahlborg, A.J., 1989a. Marked alterations in retinoid homeostasis of Sprague – Dawley rats induced by a single i.p. dose of 10 mg/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology 58, 267 – 283. Brouwer, A., Reijnders, P.J.H., Koeman, J.H., 1989b. Polychlorinated biphenyl (PCB)-contaminated fish induces vitamin A and thyroid hormone deficiency in the common seal (Phoca 6itulina). Aquat. Toxicol. 15, 99 – 106. Foster, W.G., 1995. The reproductive toxicology of Great Lakes contaminants. Environ. Health Perspect. 103 (Suppl.9), 63 – 69. Giguere, V., Onge, E.S., Segui, P., Evans, R.M., 1987. Identification of a receptor for the morphogen retinoic acid. Nature 330, 624 – 629. Hakansson, H., Manzoor, E., Trossvik, C., Ahlborg, U.G., Chu, I., Villeneuve, D., 1994. Effect on tissue vitamin A levels in the rat following subchronic exposure to four individual PCB congeners (IUPAC 77, 118, 126 and 153). Chemosphere 29, 2309 – 2313. Horton, W.E., Yamada, Y., Hassell, J.R., 1987. Retinoic acid rapidly reduces cartilage matrix synthesis by altering gene transcription in chondrocytes. Dev. Biol. 123, 508 – 516. Hynes, R.O., Yamada, K.M., 1982. Fibronectins: multifunctional modular glycoproteins. J. Cell Biol. 95, 369 – 377.
M.G. Miller et al. / Aquatic Toxicology 48 (2000) 119–125 Jensen, R.K., Zile, M.H., 1988. Effect of dietary retinoic acid on circulatory vitamin A homeostasis in polybrominated biphenyl-treated rats. J. Nutr. 118, 416–419. Kim, H.-Y., Wolf, G., 1987. Vitamin A deficiency alters genomic expression for fibronectin in liver and hepatocytes. J. Biol. Chem. 262, 365–371. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lee, L.E.J., Bols, N.C., 1991. Identification of fibronectin in rainbow trout and influence of cortisol on fibronectin accumulation by rainbow trout cell cultures. J. Aquat. Anim. Health 3, 31 – 40. Mosher, D.F., 1984. Physiology of fibronectin. Ann. Rev. Med. 35, 561 – 575. Munkittrick, K.R., Dixon, D.G., 1989. Use of white sucker (Catostomus commersoni ) populations to assess the health of aquatic ecosystems exposed to low level contaminant stress. Can. J. Fish. Aquat. Sci. 46, 1455–1462. Munkittrick, K.R., McMaster, M.E., Portt, C.B., Van Der Kraak, G.J., Smith, I.R., Dixon, D.G., 1992. Changes in maturity, plasma sex steroid levels, hepatic mixed-function oxygenase activity, and the presence of external lesions in lake whitefish (Coregonus clupeaformis) exposed to bleached kraft mill effluent. Can. J. Fish. Aquat. Sci. 49, 1560 – 1569. Napoli, J.L., Race, K.R., 1990. Microsomes convert retinol into retinoic acid and interfere in the conversions catalyzed by cytosol. Biochim. Biophys. Acta 1034, 228–232. Palace, V.P., Brown, S.B., 1994. HPLC determination of tocopherol, retinol, dehydroretinol and retinyl palmitate in tissues of lake char (Sal6elinus namaycush) exposed to coplanar 3,3%,4,4%,5-pentachlorobiphenyl. Environ. Toxicol. Chem. 3, 473 – 476. Sadaghiani, B., Crawford, B.J., Vielkind, J.R., 1994. Changes in the distribution of extracellular matrix components dur-
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ing neural crest development in Xiphophorus spp. embryos. Can. J. Zool. 72, 1340 – 1353. Scheidl, H., Chandra, T., Gmeiner, B., Zerlauth, G., Scita, G., Wolf, G., 1991. The effect of diacylglycerols on fibronectin release and its reversal by retinoic acid in cell culture. Carcinogenesis 12, 1791 – 1794. Scita, G., Wolf, G., 1994. Retinoic acid and beta-carotene inhibit fibronectin synthesis and release by fibroblasts; antagonism to phorbol ester. Carcinogenesis 15, 1043 – 1048. Scita, G., Darwiche, N., Greenwald, E., Rosenberg, M., Politi, K., DeLuca, L.M., 1996. Retinoic acid down-regulation of fibronectin and retinoic acid receptor-proteins in NIH-3T3 cells. J. Biol. Chem. 271, 6502 – 6508. Shanker, G., Sawhney, R., 1996. Retinoic acid: identification of specific receptors through which it may mediate transcriptional regulation of fibronectin gene in bovine lens epithelial cells. Cell Biol. Int. 20, 613 – 619. Spear, P.A., Bourbonnais, D.H., Norstrom, R.J., Moon, T.W., 1990. Yolk retinoids (vitamin A) in eggs of the herring gull and correlations with polychlorinated dibenzop-dioxins and dibenzofurans. Environ. Toxicol. Chem. 9, 1053 – 1061. Wolf, G., 1990. Recent progress in vitamin A research: nuclear retinoic acid receptors and their interaction with gene elements. J. Nutr. Biochem. 1, 284 – 289. Yamada, K.M., 1991. Fibronectin and other interactive glycoproteins. In: Hay, E.D. (Ed.), Cell Biology of the Extracellular Matrix, 2nd edition. Plenum Press, New York, pp. 23 – 36. Zerlauth, G., Wolf, G., 1984. Kinetics of fibronectin release from fibroblasts in response to 12-O-tetradecanoylphorbol13-acetate and retinoic acid. Carcinogenesis 5, 863 – 868. Zile, M.H., 1992. Vitamin A homeostasis endangered by environmental pollutants. Proc. Soc. Exp. Biol. Med. 201, 141 – 153.
Down-regulation of fibronectin in rainbow trout gonadal cells exposed to retinoic acid M.G. Miller a, C.M. Kapron a, C.D. Metcalfe b,*, L.E.J. Lee c,1 a Department of Biology, Trent Uni6ersity, Peterborough, Ont., Canada K9J 7B8 En6ironmental and Resource Studies Program, Trent Uni6ersity, Peterborough, Ont., Canada K9J 7B8 c Department of Veterinary Anatomy, Western College of Veterinary Medicine, Uni6ersity of Saskatchewan, Saskatoon, Sask., Canada S7N 5B4 b
Received 19 February 1999; received in revised form 3 June 1999; accepted 11 June 1999
Abstract Exposure of fish to some environmental contaminants results in alterations to the levels of retinoid (Vitamin A) stores, which could result in an increase in cellular concentrations of biologically active metabolites such as retinoic acid (RA). However, a link has not been established between changes in retinoid metabolism and impacts on the health of biota. In vitro studies with mammalian cells have demonstrated a relationship between exposure to RA and expression of the extracellular matrix protein, fibronectin (FN); a protein critical for cell migration, adhesion, and transformation. In this study, in vitro exposures of rainbow trout gonadal cells (RTG-2) to RA reduced levels of FN in culture medium; as measured using SDS-PAGE and immunoblot analysis with antisera prepared against RTG-2 cellular fibronectin. This apparent down-regulation of FN secretion occurred in a dose-dependent manner over a range of RA concentrations (10 − 10 –10 − 6 M). FN down-regulation was not accompanied by changes in the morphology of RTG-2. Future studies should be directed at determining the relationships between retinoid metabolism and FN expression and the potential effects of contaminant-induced changes to vitamin A metabolism on the health of fish. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Fibronectin; Rainbow trout; Retinoic acid
1. Introduction Fish from contaminated aquatic environments are often in poor health, as indicated by reduced growth, reproductive failure, immunosuppression, * Corresponding author. Tel.: 1-705-748-1272; fax: + 1-705748-1569. 1 Current address: Department of Biology, Wilfrid Laurier University, Waterloo, Ont., Canada N2L 3C5.
skin lesions, deformities, altered hormone levels, changes to the activity of metabolic enzymes and altered concentrations of vitamins (Munkittrick and Dixon, 1989; Arkoosh et al., 1991; Munkittrick et al., 1992; Branchaud et al., 1995; Foster, 1995; Arcand-Hoy and Metcalfe, 1999). Several investigators have observed alterations to the homeostasis of Vitamin A compounds (i.e. retinoids) in fish from areas contaminated with
0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 9 9 ) 0 0 0 5 2 - 1
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persistent organic contaminants. Branchaud et al. (1995) observed reduced levels of hepatic retinol and retinyl palmitate in white suckers (Catostomus commersoni ) sampled near urban areas of the St. Lawrence River. Malformations and eye deformities were more prevalent in sucker populations from contaminated sites. In a recent study of brown bullhead (Ameiurus nebulosus) from various sites in the lower Great Lakes, Arcand-Hoy and Metcalfe (1999) observed reduced concentrations of hepatic retinyl palmitate in bullheads from contaminated sites. Bullheads from contaminated areas had high prevalences of skin lesions. These field data are consistent with a hypothesis that retinoid homeostasis is altered in fish exposed to environmental contaminants. Several laboratory studies have examined the impacts of halogenated aromatic hydrocarbons (HAHs) such as polyhalogenated dioxins and biphenyls on vitamin A homeostasis in rodents (Jensen and Zile, 1988; Brouwer et al., 1989a; Hakansson et al., 1994). In studies with seals (Phoca 6itulina) fed PCB-contaminated fish from the Wadden Sea, Brouwer et al. (1989b) detected reduced plasma retinol levels. Palace and Brown (1994) exposed lake trout to 3,3%,4,4%,5-pentachlorobiphenyl and observed reduced concentrations of retinol, dehydroretinol and retinyl palmitate in the liver and elevated retinyl palmitate in the kidney. These studies support the hypothesis that exposure to persistent organic contaminants, and in particular HAHs, results in a decrease in retinoid stores and an increase in excretion of vitamin A catabolites (Jensen and Zile, 1988). Retinoids are essential to a variety of physiological processes, including growth, maintenance of epithelial tissues and vision, and regulation of embryogenesis and spermatogenesis (Zile, 1992). Alterations in retinoid homeostasis can induce many effects; including growth retardation, teratogenesis, epidermal lesions, ocular changes and sterility (Bhagavan, 1992). Similarities between these toxic symptoms and some of the health effects observed in fish from contaminated environments are evident; supporting the hypothesis that some of these health effects are induced through mechanisms that involve alterations to Vitamin A homeostasis.
Although exposure to HAHs appears to alter retinoid metabolism, there is no clear mechanistic link between retinoid modulation and specific toxic effects in organisms. Recent in vitro studies have shown that retinoid metabolites can induce changes in the cellular production of fibronection (FN) in mammalian cells (Scita and Wolf, 1994; Scita et al., 1996); suggesting a link between retinoid homeostasis and maintenance of this extracellular matrix protein. Through various molecular binding interactions, FN is involved in a range of cellular processes; including cell adhesion, migration, transformation and differentiation (Mosher, 1984). Thus, alterations to FN levels could have profound effects upon embryonic development, cellular differentiation and maintenance of tissues. For instance, immunohistochemical analysis has shown that FN deposition is increased along the pathways of neural crest cell migration in fish embryos; leading to speculation that FN may be involved in both the initiation and guidance of neural crest cells during their migration (Sadaghiani et al., 1994). FN is also involved in the wound healing process, as it is a component of the initial fibrin matrix deposited during blood clotting (Mosher, 1984). Several in vitro studies with mammalian and avian cells have linked exposure of cells to retinoic acid (RA) with changes in the production of cellular FN (Zerlauth and Wolf, 1984; Kim and Wolf, 1987; Horton et al., 1987; Scheidl et al., 1991; Scita and Wolf, 1994; Scita et al., 1996). Although transcription of the gene for FN is regulated by binding of RA to RA responsive elements (Wolf, 1990), these in vitro studies have shown that the direction of regulation appears to be dependent on the cell type studied. Work by Shanker and Sawhney (1996) with primary cultures of bovine lens epithelial cells showed that RA up-regulated FN by increasing transcription. Horton et al. (1987) observed that FN was upregulated in chick chondrocytes by treatment with RA, and that up-regulation was accompanied by flattening and elongation of chondrocytes so that they resembled fibroblastic cells. In all in vitro studies with mammalian fibroblasts, FN was down-regulated in a dose-dependent manner by exposure to exogenous RA (Zerlauth and Wolf, 1984; Scheidl et al., 1991; Scita and Wolf, 1994).
M.G. Miller et al. / Aquatic Toxicology 48 (2000) 119–125
In this study, in vitro assays were conducted to determine if RA can alter FN production in fish cells. The rainbow trout gonadal cell line (RTG-2) was chosen as an in vitro model because it is fibroblastic in morphology and produces large quantities of FN (Lee and Bols, 1991). RTG-2 cultures were exposed to varying concentrations of RA in serum-free medium and the medium was analyzed for FN levels using SDS-PAGE electrophoresis, followed by immunoblot analysis with antibodies raised against RTG-2 fibronectin to confirm the identity of the putative FN band. Based on studies with mammalian fibroblasts (Scita and Wolf, 1994; Scita et al., 1996), it was hypothesized that RA would down-regulate FN in a dose-dependent manner. This study is the first step in determining whether there may be a link between contaminant-induced alterations to retinoid homeostasis and health effects in fish that may be related to alterations to the extracellular matrix.
2. Methods
2.1. Cell culture and exposure to RA RTG-2 cells were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in 25 or 75 cm2 polystyrene flasks in Leibovitz’s L-15 medium (Gibco-BRL) with 10% fetal bovine serum (HyClone) and penicillin/streptomycin antibiotic mixture (100 U/ml penicillin: 100 mg/ml streptomycin). Cells were cultured in the dark at 22 9 2°C in an atmosphere of air, and were grown to confluency with fluid renewal each week. For RA exposures, 3× 105 cells were seeded into 75 cm2 flasks and then grown to confluency. Confluent monolayers were rinsed 3 times with serum-free L-15 medium to remove serum proteins, and 10 ml of serum-free medium was added that contained one of 10 − 5, 10 − 6, 10 − 7, 10 − 8, 10 − 9, and 10 − 10 M RA (Sigma) in dimethylsulfoxide (DMSO) or DMSO carrier (dissolved in media to 0.0001% v/v). Cells were maintained in this medium for 5 days prior to analysis of FN.
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2.2. SDS-PAGE analysis of FN The medium was collected from the cultures and centrifuged for 5 min with a benchtop clinical centrifuge to remove cellular debris. Solutions were then concentrated using Ultrafree-15 centrifugation filters (Millipore), which were centrifuged for 1 h at 2000× g at room temperature (22°C). The concentrated medium was pipetted out of the filter and either used immediately or stored at − 80°C for later analysis. Total protein concentration in the concentrated medium was determined using a Bio-Rad protein microassay. Samples were equalized for total protein and 10 ml subsamples were electrophoresed under reducing conditions (5% bmercaptoethanol) on 6% or 7.5% SDS-polyacrylamide gels, according to the method of Laemmli (1970). The relative mobilities of resolved proteins were compared to those of Kaleidoscope prestained molecular weight markers (BioRad). Following electrophoresis, gels were silver stained to visualize protein bands using the procedure of Blum et al. (1987); modified because of high protein levels by eliminating the final step of the staining procedure. Stained gels were scanned using a Canon DeskJet III Scanner, and scanned images were saved as .tif format files. The uncalibrated optical densities of bands were measured by analysis of.tif files using a shareware program, NIH-Image 1.56 (NIH, Bethesda, MD). Putative FN bands were identified by their position at 220 kDa relative to molecular weight markers. The relative densities of these bands were corrected for between-lane variation by dividing by the density of a reference band common to each lane and consistent in density across all lanes. Note that loadings on all lanes were equalized for protein content.
2.3. Immunodetection of FN To positively identify putative FN bands, samples were subjected to immunoblot analysis using a rabbit anti-trout FN polyclonal antibody. Antibody was prepared by purifying fibronectin from conditioned media of RTG-2 as described by Lee
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and Bols (1991). Briefly, the conditioned media of RTG-2 that had been grown in the absence of serum was collected and subjected to gelatin affinity chromatography. Column eluates were monitored for protein content and the putative fibronectin fraction was collected, dialyzed and tested for biological activity in terms of cell attachment (Lee and Bols, 1991). This preparation was then subjected to preparative SDS gel electrophoresis. A prominent doublet band was noted at 220 kDa which was eluted out of the gels using a BioRad electroeluter following manufacturer’s instructions. The eluted protein was dissolved in physiological saline and a schedule of injections were performed in rabbits following standard operating procedures for antisera production at the Animal Care Unit of the Western College of Veterinary Medicine, University of Saskatchewan. Anti-cellular fibronectin was obtained which recognized trout fibronectin as well as various commercially purchased mammalian fibronectins.
After SDS-PAGE of medium as described above, proteins were transferred in buffer (25 mM Tris; 192 mM glycine; 20% MeOH v/v) onto PVDF membranes (Immobilon-P, Millipore) on a Bio-Rad electroblot apparatus (100 V constant for 1 h). The membranes were blocked in a 7.5% purified non-fat milk solution (Bio-Rad) for 30 min at 37°C. Rabbit anti-trout FN antibodies at a dilution of 1:1000 (in 5% milk) were applied for 1 h at room temperature with agitation, followed by three 10 min washes in TBS-T (20 mM Tris, 137 mM NaCl, 1% v/v Tween-20, pH 7.6) under the same conditions. The blots were then incubated with horseradish-peroxidase labelled secondary antibodies (goat anti-rabbit IgG1, Jackson Immunoresearch Labs) at 1:5000 dilution in a 5% milk/TBS-T solution. The membranes were then washed (3× 10 min) in TBS-T and proteins were detected using the ECL Western Blotting Detection system (Amersham).
3. Results and discussion
Fig. 1. Polyacrylamide gels (silver stained) showing the putative fibronectin (FN) band prepared from samples of rainbow trout gonadal cells (RTG-2) exposed to retinoic acid (RA) at concentrations of 10 − 8, 10 − 7 and 10 − 6 M. Also indicated are the molecular weight (MW) markers at 71, 133 and 202 kDa and the reference band used in the semi-quantitative analysis of optical densities of the putative FN band.
Exposure of RTG-2 to RA resulted in a decrease in the density of the putative FN bands; especially at exposure concentrations \10 − 8 M (Fig. 1). As illustrated in Fig. 2, the decline in density of the bands was concentration dependent; fitting a regression line of y= − 0.050 ln x− 0.088 (r 2 = 0.92). Immunoblot analysis of culture media confirmed the tentative identification of the FN band in earlier experiments, and also showed a dose-dependent decrease in FN over a concentration range of 10 − 8 –10 − 6 M RA (Fig. 3). This response is consistent with down-regulation of FN secretion into culture media in response to increasing RA concentrations. RTG-2 in culture appeared fibroblastic in morphology; characterized by a flattened appearance with multiple protrusions of the cytoplasm. In culture, the cells grew from several foci on the dish in a spiral pattern. In treatments with RA, there were no differences in density or attachment of RTG-2, and there was no alteration to the morphology of the cells. In mammalian chondrocytes, FN up-regulation was accompanied by dis-
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Fig. 2. Relative optical density of putative fibronectin (FN) bands in polyacrylamide gels (silver stained) using samples prepared from rainbow trout gonadal cells (RTG-2) in control treatment and in treatments with retinoic acid (RA) at concentrations of between 10 − 9 and 10 − 5 M. The arrow on the y-axis indicates the optical density of the band from the control treatment.
Fig. 3. Representative immunoblot of polyacrylamide gels showing fibronectin (FN) bands identified by immunoblot analysis with rabbit anti-trout FN polyclonal antisera in samples prepared from rainbow trout gonadal cells (RTG-2) exposed to various concentrations (10 − 10 –10 − 8 M) of retinoic acid (RA).
tinct flattening of cells to a more fibroblastic form (Horton et al., 1987); consistent with the role of the extracellular matrix, and FN in particular, in regulating cell morphology (Hynes and Yamada, 1982). The lack of a change in morphology in RTG-2 in treatments where RA-induced downregulation of FN was observed may indicate that the residual extracellular matrix (ECM) proteins remaining from pre-treatment continued to support the normal morphology of the cells through-
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out the 5 day exposure period. Longer exposures to RA may induce changes in the morphology of RTG-2. While the kinetics of release of FN and ECM proteins is well known for other cell types (Scita et al., 1996), RTG-2 may not follow the same pattern as mammalian cell lines. These results with RTG-2 are consistent with the down-regulation of FN observed in in vitro studies with mammalian fibroblasts (Scita and Wolf, 1994; Scita et al., 1996), but are contrary to other studies that show RA-induced up-regulation of FN in chick chondrocytes (Horton et al., 1987) and bovine lens epithelial cells (Shanker and Sawhney, 1996). The reasons for the differences between cell types is not known, but may be associated with differences in FN gene expression following binding of RA with RA responsive elements (Shanker and Sawhney, 1996). It appears from this study that RTG-2 resemble mammalian fibroblasts in the pattern of regulation of FN expression. This study also illustrates the potential for using RTG-2 as an in vitro model for studies of the effects of retinoids on gonadal differentiation and development. It is difficult to interpret these in vitro results with RTG-2 in terms of potential in vivo responses in fish. Different tissues may vary in RA-induced regulation of FN expression. In addition, RA is rapidly metabolized in vivo because of the potent biological effects of this retinoid compound at various locations in the body (Blomhoff et al., 1991). For instance, as a morphogen during embryonic development, RA is transiently maintained at specific target tissues such as limb buds to control positional morphology (Giguere et al., 1987). Therefore, one needs to know more about the metabolism of retinoids in fish in order to determine if RA is mobilized in response to reduced levels of the storage forms of retinoids and whether RA is ‘delivered’ to tissues in concentrations sufficient to alter FN expression. While RA concentrations in human plasma are approximately 10 − 9 M (Bhagavan, 1992), the endogenous concentrations of RA in fish plasma and tissues have not been studied. Finally, FN occurs in several different forms that vary between species and even within a single organism; differing in structural features such as binding sites and
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glycosylation locations (Yamada, 1991). There may be differences in responses to RA that are related to structural differences in FN. Field studies have shown that retinoids are mobilized in fish exposed to environmental contaminants (Branchaud et al., 1995; Arcand-Hoy and Metcalfe, 1999), and this has been supported by experimental studies with fish exposed to PCB compounds (Palace and Brown, 1994). In birds and mammals exposed to HAHs, mobilization of retinoid stores in the liver occurs through metabolism of non-polar (i.e. fat-soluble) retinyl esters to more polar retinol (Spear et al., 1990; Zile, 1992). It is not clear whether higher levels of serum retinol can lead to altered concentrations of cellular RA. However, in vitro studies with rat liver microsomes showed that both retinol and retinal are metabolically converted into retinoic acid (Napoli and Race, 1990). Considerablely more work is required to determine whether there is a possible link between contaminant-induced mobilization of retinoid stores in organisms and changes to cellular RA levels that could have potent biological effects.
4. Conclusions In this study, RA appeared to down regulate FN in a dose-dependent manner in rainbow trout gonadal cells. This molecular response was not accompanied by changes in cell morphology under the in vitro conditions used in this study. More work is required to determine if these responses can be observed in vivo in fish species, and to determine whether RA-induced alterations in FN expression are linked to specific health effects in fish. Further study is also needed to characterize changes to vitamin A metabolism (including cellular production of RA) caused by exposure of fish and other biota to environmental contaminants.
Acknowledgements Katharine Haberstroh helped with laboratory procedures. This work was supported by Research
Grants from the Natural Sciences and Engineering Research Council to Chris Metcalfe and to Carolyn Kapron.
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