Differential cell sensitivity to cadmium exposure in RTgill-W1, RTG-2, and RTL-W1 rainbow trout (Oncorhynchus mykiss) cell lines: an in vitro cell line model to study cadmium-induced cytotoxicity.
Cadmium is intimately connected to Idaho through mining practices and other emissions of this toxic metal into the environment. Since aquatic organisms can serve as bioindicators to assess the possible impact of heavy metal contaminants, we used liver (RTL-W1), gonadal (RTG-2), and gill (RTgill-W1) cell lines derived from rainbow trout to investigate cadmium-induced cytotoxicity. We hypothesize RTgill-W1 cells, which are frequently exposed to heavy metal contaminants, are more resistant to cadmium compared to RTL-W1 or RTG-2 cells. Cells were exposed to 200 [mu]M or 500 [mu]M cadmium chloride (Cd[Cl.sub.2]) for 0, 3, 12, 24 or 48 hours. Parameters assessed were cell morphology by phase-contrast microscopy, loss of adherence, and viability using a trypan blue exclusion assay. RTgill-W1 cells were more resistant to 200 [micro]M Cd[Cl.sub.2] compared to RTL-W1 and RTG-2 cells. When exposed to 500 [micro]M Cd[Cl.sub.2], RTgill-W1 remained more resistant (29.2 % viability), followed by RTG-2 (8.4% viability), and RTL-W1 cells (1.0% viability) at 48 hours. For comparison, we examined the effect of cadmium exposure on primary gill cells isolated from live rainbow trout to the RTgill-W1 cell line. There was no difference in viability between RTgill-W1 and primary gill cells exposed to 200 [micro]M Cd[Cl.sub.2]. When exposed to 500 [mu]M Cd[Cl.sub.2], primary gill cells were more resistant compared to RTgill-W1 cells. Collectively, RTgill-W1 and primary gill cells are more resistant to cadmium than RTL-W1 or RTG-2. This differential sensitivity to cadmium provides a model for elucidating the intracellular mechanisms by which cells respond to and protect against cadmium's toxic action. Furthermore, this research illustrates the importance of using multiple cell lines to access cytotoxicity in response to heavy metal contaminants.
KEYWORDS: Cadmium, rainbow trout, RTgill-W1, RTG-2, RTL-W1
Cadmium is a widespread heavy metal contaminant that poses a serious threat to the environment and human health (CDC, 2005; Gusmao Lima et al., 2006). Cadmium emission into the environment occurs through use of fertilizers, plastic stablizers, electroplating, mining practices, and the discard of electronic products, such as cell phones and computers, into landfills (Thornton, 1992; Chien et al. 2003; Jarup, 2003; Rydh & Svard, 2003; EPA 2003; CDC, 2005). This toxic metal is a known mutagen, teratogen, and carcinogen (Degrave, 1981; Slebos et al., 2006). Human exposure to cadmium occurs through ingestion of contaminated food or water, or by smoking cigarettes (CDC, 2005). Exposure to cadmium is associated with lung, kidney, bone, and liver disease, and various cancers (Latinowo et al., 1997; Jarup et al., 1998; Waisberg et al., 2003; Jarup & Alfven, 2004; Nordberg et al., 2004; Jin et al., 2004a; Jin et al., 2004b).
Aquatic organisms serve as biological indicators of the possible impacts of environmental toxins, such as cadmium, on human health (Dayeh et al., 2002; Caminada et al., 2006). Waterborne and dietary cadmium accumulates in fish kidney, gill, liver, and gonadal tissue leading to deleterious effects (Burger et al., 2004; Dural et al., 2006). In the present study, we selected a cell line model derived from rainbow trout. Fish cell lines and primary cell cultures are increasingly used in research to investigate the effects of heavy metals (Risso-de Faverney et. al., 2001; Mazon et al., 2004; Dayeh et al., 2005; Caminada et al., 2006). Studies indicate that fish have similar intracellular mechanisms to mammals such as oxidative stress, apoptosis, and defense mechanisms in response to heavy metal contaminants (Kelly et al., 1998; Risso-de Faverney et al., 2001). However, the effects of cadmium exposure on these intracellular mechanisms in fish are relatively unexplored (Bols et al., 2005). Furthermore, fish routinely encounter heavy metal contaminants and may have developed novel cellular mechanisms for protection.
In the current study, we used liver (RTL-W1), gonadal (RTG-2), and gill (RTgill-W1) cell lines derived from rainbow trout to investigate cadmium-induced cytotoxicity. We hypothesize that gill cells are more resistant to cadmium exposure compared to liver or gonadal cells because they are frequently exposed to environmental contaminants. To test this hypothesis, our research aims to establish a cytotoxicity profile for the three rainbow trout cell lines. A long term goal is to use rainbow trout cell lines as an in vitro model to study cadmium's action on a cellular and molecular level.
MATERIALS AND METHODS
Fish Cell Lines
RTG-2 and RTgill-W1 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). RTL-W1 cell line was kindly donated by Dr. Niels Bols at the University of Waterloo, Canada. Cells were cultured in flasks containing Leibovitz's L-15 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (ATCC, Manassas, VA), 100 IU/ml penicillin, and 100[micro]g/ml streptomycin (Sigma-Aldrich, St. Louis, MO) at 19[degrees]C (RTgill-W1 and RTL-W1) or 21[degrees]C (RTG-2). For routine maintenance, medium was changed every 3 to 4 days and cells were subcultured weekly.
Primary Gill Cells
Live rainbow trout were generously donated by the Nampa Fish Hatchery, Nampa, ID. During transport to The College of Idaho, fish were kept in an aerated cooler. Upon arrival, fish were sacrificed by blunt trauma to the head and gill cells were collected as previously described (Part, 1993). Briefly, gill arches were removed and gill filaments were collected and placed in sterile phosphate buffered saline (PBS) supplemented with 100 IU/ml penicillin, 100[micro]g/ml streptomycin, 2mM glutamine, and 2.5 [micro]g/ml fungizone (Sigma-Aldrich, St. Louis, MO). The gill filaments were washed six times and dissociated in 0.05% trypsin and 0.02% EDTA. The cells were collected by filter through 80 [micro]m mesh into sterile PBS supplemented with 10% fetal bovine serum. The filtration process was repeated four times and the cells were collected by centrifugation for 10 min. at 200 x G. Cells were plated in 6-well culture plates at a density of 5 x [10.sup.6] cells/well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 [micro]g/ml streptomycin, 2mM glutamine, and 2.5 [micro]g/ml fungizone for 24 hours at 18[degrees]C prior to the addition of Cd[Cl.sub.2] (Sigma-Aldrich, St. Louis, MO).
Cells were plated in 6-well culture plates at 3 x [10.sup.5] cells/well (cell lines) or 5 x [10.sup.6] cells/well and treated with 200 or 500 [micro]M Cd[Cl.sub.2] (micromoles of Cd[Cl.sub.2] per liter of medium) for 0, 3, 12, 24 or 48 hours. Controls received medium only. The concentrations of Cd[Cl.sub.2] used in this study are within the range reported for in vitro systems (Lyons-Alcantara et al, 1998; Sokolova et al, 2004; Dayeh et al, 2005). The Cd[Cl.sub.2] concentration range is high in comparison to quantities found in the environment but necessary to induce an acute response in an in vitro culture system.
Cells were grown on cover slips in 6-well culture plates at 3 x [10.sup.5] cells/ well and treated with 200[micro]M Cd[Cl.sub.2] for 0, 24, or 48 hours. Cell morphology was assessed by phase-contrast microscopy using a Nikon epifluorescence eclipse E400 microscope. Digital images were captured using ImagePro Express by Media Cybernetics (Silver Spring, MD).
Trypan Blue Exclusion Assay
After treatment, the supernatant was collected and a cell count was conducted using a hemacytometer and light microscopy to determine loss of cell adherence. The adherent cells were removed using 0.25% trypsin-EDTA. All cells were stained with 0.05% trypan blue and counted. Cells with an intact cell membrane did not uptake the trypan blue dye and were considered viable.
Data represents the mean [+ or -] standard deviation (SD) or standard error (SEM) of at least three separate experiments. Data were analyzed using a one-way analysis of variance followed by a Tukey test for multiple comparisons, or by a Student's t-test for comparison between two groups. A p-value < 0.05 was considered significant.
[FIGURE 1 OMITTED]
Treatment with 200 [micro]M Cd[Cl.sub.2] for 24 or 48 hours resulted in no observable change in RTgill-W1 cell morphology compared to the untreated control (Figure 1A). In contrast, distinct morphological changes were observed in RTL-W1 cells treated with 200 [micro]M Cd[Cl.sub.2] for 48 hours. RTL-W1 cells appeared more rounded in shape, smaller in size, and detached from the culture plate when exposed to Cd[Cl.sub.2] (Figure 1 B). To quantify this observation, we assessed the effect of Cd[Cl.sub.2] on loss of cell adherence.
There was no loss of adherence observed at any time point in RTgill-W1 cells treated with 200 [micro]M Cd[Cl.sub.2] However, RTL-W1 cells treated with 200 [micro]M Cd[Cl.sub.2] resulted in a significant loss of adherence at 48 hours (Figure 2A). These data are consistent with the cell morphology assessment. Due to the marked difference in response to cadmium exposure between RTgill-W1 and RTL-W1 cells, we introduced a third cell line, RTG-2, to our research. Similar to RTL-W1, there was a significant loss of adherence observed in RTG-2 cells treated with 200 [micro]M Cd[Cl.sub.2]at 48 hours (Figure 2A).
A trypan blue exclusion assay was used to determine whether cell viability differed between the cell lines in response to cadmium exposure. There was no change in RTgill-W1 cell viability in response to 200 [micro]M Cd[Cl.sub.2] at any time point (Figure 2B). In contrast, RTL-W1 and RTG-2 cells exhibited a significant decrease in cell viability when exposed for 48 hours to 200 [micro]M Cd[Cl.sub.2] (Figure 2B). When differences were analyzed between cell lines, loss of adherence and trypan blue data indicated RTgill-W1 cells were more resistant to 200 [micro]M Cd[Cl.sub.2] compared to RTL-W1 and RTG-2 cells at 48 hours.
RTL-W1 and RTG-2 cells showed a significant loss of adherence by 48 hours with 500 [micro]M Cd[Cl.sub.2] treatment. When differences were analyzed between cell lines, loss of cell adherence was significantly greater in RTL-W1 and RTG-2 cells compared to RTgill-W1 cells exposed to 500 [micro]M Cd[Cl.sub.2] (Figure 3A). RTgill-W1 cells were the most resistant with 29.2% viability, followed by RTG-2 cells with 8.4% viability, and RTL-W1 cells with less than 1.0% viable cells remaining at 48 hours with 500 [micro]M Cd[Cl.sub.2] treatment (Figure 3B). Collectively, RTgill-W1 cells are more resistant to cadmium compared to RTG-2 and RTL-W1 cells.
[FIGURE 2 OMITTED]
Next, we examined the effect of cadmium exposure on cell viability using gill cells derived from live rainbow trout compared to the RTgill-W1 cell line. There was no significant difference between RTgill-W1 and primary gill cells treated with 200 [micro]M Cd[Cl.sub.2] (Table 1). Primary gill cells exposed to 500 [micro]M Cd[Cl.sub.2] exhibited more resistance compared to the RTgill-W1 cell line (Table 2). However, both RTgill-W1 and primary gill cells were more resistant to Cd[Cl.sub.2] compared to RTL-W1 cells (Table 2).
[FIGURE 3 OMITTED]
This research supports the hypothesis that RTgill-W1 cells are more resistant to cadmium exposure compared to RTL-W1 and RTG-2 cells. Other studies using cytotoxicity assays demonstrate cell lines derived from rainbow trout respond differently to environmental contaminants including heavy metals (Dayeh et al., 2002; Dayeh et al., 2005; Caminada et al., 2006). Treatment with 200 [micro]M Cd[Cl.sub.2] resulted in no morphological change, loss of adherence, or decrease in viability in RTgill-W1 cells. In contrast, RTL-W1 cells treated with 200 [micro]M Cd[Cl.sub.2] resulted in observable changes in morphology. Both RTL-W1 and RTG-2 cells exhibited a significant loss in adherence and decrease in viability in response to 200 [micro]M Cd[Cl.sub.2]. When exposed to 500 [micro]M Cd[Cl.sub.2], RTgill-W1 remained more resistant (29.2 % viability), followed by RTG-2 (8.4% viability), and RTL-W1 cells (1.0% viability) at 48 hours. For comparison, we examined the effect of cadmium exposure on primary gill cells isolated from live rainbow trout to the RTgill-W1 cell line. There was no difference in cell viability between RTgill-W1 and primary gill cells exposed to 200 [micro]M Cd[Cl.sub.2]. When the concentration was increased to 500 [micro]M Cd[Cl.sub.2]. primary gill cells were more resistant (as determined by percent viable cells) compared to RTgill-W1 cells; however, both gill cultures were more resistant to cadmium compared to RTL-W1 cells. Together, these data suggest that the response to cadmium is more similar between RTgill-W1 and primary cultured gill cells than to RTL-W1 cells. The difference between the two gill cultures in response to 500 [micro]M Cd[Cl.sub.2] may be attributed to the cellular heterogeneity of the primary gill cell culture which could contribute to a greater resistance at higher concentrations of cadmium (Bols et al., 2005). Collectively, these results indicate rainbow trout cell lines respond differently to cadmium exposure and therefore could be used as an experimental model to study the underlying mechanisms involved in cell-specific cytotoxic effects of heavy metals.
Our research identifies rainbow trout cell lines as a useful model to study the mechanisms that protect against cadmium toxicity. One explanation for why RTgill-W1 cells are more resistant to cadmium is through induction of protective mechanisms that reduce the toxic action of cadmium. Metallothionein (MT), a protein known to sequester metals within a cell, is induced in rainbow trout gill and liver tissue in response to waterborne or dietary cadmium administration (Chowdhury et al., 2005). Furthermore, in vivo exposure to cadmium results in an increase in MT mRNA expression in rainbow trout hepatocytes and branchial tissue (Lange et al., 2002). However, an in vitro study reports no change in MT protein expression in rainbow trout epithelial cells exposed to cadmium, illustrating that cadmium's effects can be cell-specific and the method of exposure needs consideration (Lyons-Alcantara et al., 1998).
In addition to understanding the different mechanisms involved in protection against cadmium-induced cytotoxicity, this rainbow trout cell line model can be used to investigate intracellular pathways altered following exposure to cadmium. For example, rainbow trout cell lines can provide insight into the mechanisms of cell death activated in response to cadmium exposure. Cells can die by necrosis (pathologic cell death) or apoptosis, also referred to as programmed cell death (Potten & Wilson, 2004). In primary rainbow trout hepatocytes, exposure to cadmium results in DNA fragmentation and activation of caspases 3, 8, and 9, hallmarks of apoptosis (Risso-de Faverney et al., 2001; Risso-de Faverney et al., 2004). However, the effect of cell death can be concentration-dependent, with a high concentration of cadmium leading to necrosis (Lopez et al., 2003; Coonse et. al, 2007). Therefore, one possibility is that cadmium exposure induces different cell death pathways contributing to the differential cell sensitivity between the three cell lines.
Another use for the rainbow trout cell line model is to study the intracellular mechanisms implicated in cadmium-induced oxidative stress. Cadmium treatment induces oxidative stress in primary cultured rainbow trout hepatocytes through formation of reactive oxygen species that ultimately leads to apoptosis (Risso-de Faverney et al., 2001; Risso-de Faverney et al., 2004). Glutathione, an antioxidant, is depleted in cultured rainbow trout hepatocytes and RTG-2 cells exposed to cadmium which ultimately leads to metal-induced oxidative damage (Maracine et al., 1998; Risso-de Faverney et al., 2001; Risso-de Faverney et al., 2004). Therefore the resistance to cadmium observed in RTgill-W1 cells may be related to the level of antioxidants available within the cell.
In summary, since these rainbow trout cell lines exhibit differential sensitivity to cadmium exposure, they provide a model for elucidating the intracellular mechanisms by which cells respond to and protect against cadmium's toxic action. Furthermore, these data illustrate the importance of using multiple cell lines to access cytotoxicity in response to environmental toxins.
This work was supported by NIH Grant P20RR016454 from the INBRE Program of the National Center for Research Resources. The authors also appreciate the helpful advice from Kendra Coonse, the generous donation of the RTL-W1 cell line from Dr. Niels Bols, and the generosity of the Nampa Fish Hatchery, Nampa, ID.
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Department of Biology, The College of Idaho (formerly Albertson College of Idaho), 2112 Cleveland Blvd, Caldwell, ID 83605, USA
Correspondence to: S. J. Heggland; Tel.: 208-459-5063; Fax: 208-459-5044; email: sheggland@ collegeofidaho.edu
W. A. Harvey *, S.T. Frost *, K.T. Machynia, M. Gerdes, S. J. Heggland
* contributed equally to the research
Table 1. The effect of Cd[Cl.sub.2] on viability in RTgill-W1, primary gill and RTL-W1 cells. Cells were treated with 200 [micro]M Cd[Cl.sub.2] for 0, 24, or 48 hours. Following treatment, all cells were stained with trypan blue. Results are expressed as percent viable cells. Each line represents the mean [+ or -] SEM or [+ or -] SD (primary gill cells) of at least three independent experiments and 0 hour represents untreated cells. * Denotes significant difference from control p<0.05. RTgill-W1 Primary Gill RTL-W1 O Hr 94.26 [+ or -] 0.98 86.41 [+ or -] 13.67 94.71 [+ or -] 2.06 24 Hr 91.49 [+ or -] 1.01 79.13 [+ or -] 11.06 87.48 [+ or -] 1.72 48 Hr 92.37 [+ or -] 1.68 86.1 [+ or -] 2.42 71.1 [+ or -] 5.48 Table 2. The effect of Cd[Cl.sub.2] on viability in RTgill-W1, primary gill and RTL-W1 cells. Cells were treated with 500 [micro]M Cd[Cl.sub.2] for 0, 24, or 48 hours. Following treatment, all cells were stained with trypan blue. Results are expressed as percent viable cells. Each line represents the mean [+ or -] SEM or [+ or -] SD (primary gill cells) of at least three independent experiments and 0 hour represents untreated cells. * Denotes significant difference from control p<0.05. RTgill-W1 Primary Gill RTL-W1 O Hr 92.7 [+ or -] 1.52 86.41 [+ or -] 13.67 97.86 [+ or -] 1.63 24 Hr 46.34 [+ or -] 6.38 88.93 [+ or -] 12.96 19.07 [+ or -] 7.87 48 Hr 29.28 [+ or -] 3.21 51 [+ or -] 11.66 0.84 [+ or -] 0.43
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|Author:||Harvey, W.A.; Frost, S.T.; Machynia, K.T.; Gerdes, M.; Heggland, S.J.|
|Publication:||Journal of the Idaho Academy of Science|
|Date:||Jun 1, 2008|
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