We have previously reported dose-response characteristics associated with BDE 47 toxicity in transformed rainbow trout gill and liver cells (Shao et al., 2008a
). The dose-response curves previously observed for BDE 47 were strikingly similar to other studies using mammalian primary and transformed cells exposed to either BDE 47 or to PBDE congener mixtures (Costa and Giordano, 2007
; Giordano et al., 2008
; Shao et al., 2008b
; Yu et al., 2008
), suggesting that these agents may have similar mechanisms of toxicity in fish and mammalian cells. However, the biochemical events underlying the progression of BDE 47 cell injury in fish have not been well characterized. The application of flow cytometry approaches and use of fluorescently labeled probes that sensitively detect losses of cell function provides a valuable approach to test the hypothesis that the acute toxicity of BDE 47 in trout gill cells is associated with mitochondrial oxidative injury leading to apoptosis. To our knowledge, the flow cytometry probes used in the present study have not been applied to studies using fish cells, at least in a comprehensive approach to identify toxicants that disrupt cellular redox status.
In the current study, the use of the PI, a dye that binds to double-stranded DNA, enabled us to determine the effect of BDE 47 on the percentage of gill cells in the sub G1 phase of the cell cycle while concomitantly allowing for comparison of results obtained by flow cytometry and biochemically. We conducted the two viability assays (i.e., PI and alamarBlue) under the same conditions with similar results (Shao et al., 2008a
). The BDE 47-induced loss of NADPH autofluorescence was consistent with biochemical changes associated with the loss of mitochondrial energetics using the alamarBlue reduction assay that reported in our earlier studies (Shao et al., 2008a
; Shao et al., 2008b
). Because the alamarBlue assay measures the metabolic integrity of the mitochondria based upon the function of mitochondrial reductases (Ahmed et al., 1994
), the rate of alamarBlue reduction reflects oxidation-reduction activity of respiratory chain components in mitochondria, a major intracellular source of ROS (Lee and Wei, 2007
). Therefore, our observations of decreased mitochondrial cardiolipin content and mitochondrial membrane potential on exposure to low concentrations of BDE 47 is consistent with the generation of oxidative stress within the mitochondria as a mechanism of cell injury, and indicates that mitochondria are a subcellular target of PBDEs in fish.
A close examination of the flow cytometry data reveals the nature of mitochondrial perturbations caused by BDE 47. For example, the BDE 47-induced reduction in the FS signal, and concomitant increase in the SS signal, reflects morphological characteristics associated with the onset of apoptosis. The results obtained by using the JC-1 fluorescent assay were consistent with those by MitoTracker Red indicating a loss of mitochondrial redox potential which is typically correlated with apoptosis. These data are consistent with analysis of sub G1 DNA content analysis by flow cytometry which indicated that exposure to BDE 47 increased the percentage of cells undergoing frank apoptosis. Endonucleases activated during apoptosis cleave sections of internucleosomal DNA, causing extensive DNA fragmentation, a primary indicator of apoptosis (Nagata, 2000
). As a result of DNA fragmentation and chromatin condensation, apoptotic cells show reduced DNA staining with PI, reflecting lower quantitative DNA content relative to G1 phase nuclei (Gong et al., 1994
). The shedding of apoptotic bodies containing fragments of nuclear chromatin may further contribute to the loss of DNA from apoptotic cells (Pozarowski et al., 2004
). Accordingly, the emergence of sub-G1 peaks seen with increasing levels of BDE 47 treatment indicates that these cells were undergoing apoptosis. These results are consistent with other studies in primary human fetal liver hematopoietic stem cells (Shao et al., 2008b
) and primary mouse neurons and astrocytes (Giordano et al., 2008
) which also demonstrated that exposure to PBDEs lead to cellular injury through induction of apoptotic cell death.
A substantial body of work has shown that mitochondrial membrane depolarization plays an important role in regulating apoptosis (Green and Kroemer, 2004
; Marchetti et al., 1996
). Chemical exposures can destabilize the mitochondrial membrane and cause a loss of mitochondrial integrity and cell survival (Loh et al., 2006
; Zorov et al., 2006
). In the current study, we observed a marked loss of mitochondrial membrane potential associated with the degradation of cardiolipin, a marker of mitochondrial membrane lipid oxidation, in gill cells treated with BDE 47, further supporting our hypothesis that BDE 47 targets the mitochondria and stimulates apoptosis via mitochondrial oxidative injury (Lugli et al., 2005
). Cardiolipin is a phospholipid located in the inner mitochondrial membrane, that when oxidized, plays a crucial role in cytochrome C release from mitochondria leading to apoptosis (Basova et al., 2007
). Normally, cytochrome C is localized to the outer surface of the inner mitochondrial membrane via electrostatic interactions with anionic lipids such cardiolipin (Demel et al., 1989
). BDE 47 mediated cardiolipin oxidation likely disrupted the interaction between cytochrome C and cardiolipin, followed by its dissociation from the membrane and escape through the Bax-mediated permeabilized outer mitochondrial membrane (Nomura et al., 2000
; Ott et al., 2002
; Petrosillo et al., 2001
). It is also possible that BDE 47 triggered mitochondrial swelling and a rupture of the outer mitochondrial membrane (as reflected by light scattering measurements), which could have then caused cytochrome C release (Goldstein et al., 2000
), leading to the activation of caspase-dependent and independent mitochondrial death pathways (Antonsson, 2004
It is important to note that all of the fluorescently-labeled commercial dyes used in this study required substantial optimization methodologies during assay validation. Furthermore, in addition to the battery of cell indicators discussed (i.e. JC-1, MitoTracker, NAO, PI staining, NADPH autofluorescence), we initially investigated other probes, including BODIPY FL C11, diphenyl-1-pyrenylphosphine (DPPP) and Annexin-V-Fluorescein (Annexin-V-FL). Specifically, BODIPY FL C11 is a lipophilic fluorophore that localizes to cell membranes and emits a fluorescence profile reflective of the oxidative state of the plasma membrane. Accordingly, as BODIPY FL C11 becomes oxidized, there is a concomitant shift from the red fluorescent excimer form to a green fluorescent monomeric form associated with peroxidation state of the membrane. By contrast, Annexin-V-FL is a phospholipid-binding protein with strong affinity for phophotidylserine (PS). During the early stages of apoptosis, PS is translocated from the inner part of the plasma membrane to the external surface of the cell. Thus, Annexin-V-FL binds exposed PS, providing a direct measure of apoptotic events. Although these aforementioned probes were used effectively in our studies addressing the mechanisms of BDE 47 injury to human fetal liver hematopoietic stem cells (Shao et al., 2008b
), they yielded inconsistent results in trout gill cells. The unsuccessful application of some of these flow cytometry probes may have been a result of the gill cell phospholipid content, which appears to be relatively low in trout gill cell membranes and can be further reduced during thermal acclimation (Hazel and Carpenter, 1985
; Kraffe et al., 2007
). Furthermore, the fact that the trout gill cells were maintained at 19°C, a higher than normal physiological temperature for rainbow trout, may have further decreased the content of unsaturated phospholipids allowing for detection of lipid peroxidation by BODIPY FL C11 and DPPP. The lower mitochondrial potential of rainbow trout gill cells relative to other tissues may have contributed to the inability of JC-1 to form aggregates (O’Dowd et al., 2006
). Although we were not able to capture the red JC-1 signal by flow cytometry, the observed increase of the corresponding JC-1 green signal is similar to results observed using fetal liver hematopoietic stem cells, and in the current study using the MitoTracker Red dye.
As discussed, the dosing range of BDE 47 used in this study (3.2 μM -50 μM) allowed for comparison to other studies and were reasonable given that a only a single acute dose was employed, likely underestimating the environmental scenario involving chronic exposures. This range of exposures encompassed somewhat higher BDE 47 levels that caused cytotoxicity as well as lower BDE 47 levels that elicited subtle biochemical changes that allowed us to clearly evaluate with mechanisms of toxicity. The cellular effects observed with low micromolar concentrations of BDE 47 suggests a moderate sensitivity of trout gill cells to this, and potentially to other, PBDE congeners. BDE 47 toxicity to primary hepatocytes from rainbow trout show similar sensitivities which may be partially dependent upon the poor detoxification capabilities of the cells (Nakari and Pessala, 2005
). Although the role of detoxification gene expression has not been clearly identified in detecting against PBDE toxicity, a number of genes critical to cellular protection are down-regulated in the liver of rainbow trout exposed to BDE 47 in vivo
(Hook et al., 2006
). The sensitivity of trout gill epithelial cells to BDE 47 may be due to their poor ability to detoxify reactive oxygen species via cellular antioxidant pathways (Shao et al., 2008a
). However, we did not characterize the baseline expression of antioxidant enzymes in the gill cells, or determine if BDE 47 exposure modulates the expression of genes that protect against oxidative stress. These studies are ongoing in our laboratory.
In summary, BDE 47 is a predominant PBDE congener detected in fish that causes injury to cultured rainbow trout gill cells that is associated with increased cellular oxidative stress, mitochondrial injury, and apoptosis. Although the resulting cell death occurs at a level that is above PBDE concentrations encountered in the environment, we have not examined the effect of multiple or chronic exposures which may be more relevant to environmental exposures. Furthermore, the fact that BDE 47 bioaccumulates in fish relative to the other higher molecular weight congeners (Betts, 2002
; Browne et al., 2009
; Sjodin et al., 2003
) is important in extrapolating our in vitro
results to more relevant environmental scenarios. Collectively, our studies also indicate that oxidative stress-associated biomarkers may be useful in assessing the sublethal effects of PBDEs in fish, as well as in other species. To this end, the application of flow cytometry-based analyses and fluorescent probes that are sensitive markers of cell injury from aquatic species are of utility for broader use in the field of aquatic toxicology.