* University at Albany, School of Public Health, Department of Environmental Health and Toxicology, Rensselaer, New York 12144;
New York State Department of Health, Wadsworth Center, Albany, New York 12201; and
University at Albany, Institute for Health and the Environment and School of Public Health, Department of Environmental Health and Toxicology, Rensselaer, New York 12144
Received June 9, 2003; accepted August 25, 2003
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ABSTRACT |
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Key Words: rat; mitochondria; endoplasmic reticulum; calcium; membrane potential; coplanar PCBs; cyclosporin A; lipid bilayers; mitochondrial permeability transition channel.
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INTRODUCTION |
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The immune system has long been known to be sensitive to PCBs, and is among the most sensitive of all organ systems (ATSDR, 2000). Administration of PCBs to animals causes atrophy of the thymus gland and immunosuppression, and the evidence suggests that much of this action is mediated via activation of the Ah receptor (Davis and Safe, 1990
; Tryphonas et al., 1991
; Zhao et al., 1997
). However an Ah-receptor independent mechanism has also been suggested (Denomme et al., 1983
; Holsapple et al., 1986
; Kerkvliet et al., 1990a
,b
; Schuetz et al., 1986
). Harper et al.(1993)
demonstrated splenic plaque-forming cell responses to antigen were reduced by di-ortho, highly chlorinated PCB congeners that are inactive at the Ah receptor. Smialowicz et al.(1997)
demonstrated that the di-ortho PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl), which does not activate the Ah receptor, enhanced antibody plaque-forming cell responses to sheep red blood cells, whereas TCDD resulted in a dose-dependent suppression. Similarly Schulze-Stack et al.(1999)
found that PCB137 (2,2',3,4,4',5 hexachlorobiphenyl) and PCB153, which do not activate the Ah receptor, significantly inhibited LPS-induced proliferation of splenocytes in mouse strains with either high or low Ah receptor expression. A major goal of these experiments was to determine whether we could demonstrate effects of non-dioxin-like PCBs on cells of the immune system.
While the mechanism(s) responsible for non-Ah receptor-mediated immunosuppression are not certain, Shin et al.(2000) have demonstrated that the tetra-ortho congener, PCB 104 (2,2',4,6,6'-pentachlorobiphenyl), causes apoptosis in human monocytic U937 cells, and Jeon et al.(2002)
demonstrated PCB-induced apoptosis by Aroclor 1254 as well as PCB congeners 47 (2,2',4,4'-tetrachlorobiphenyl), 52 (2,2',5,5'-tetrachlorobiphenyl), 128 (2,2',3,3',4,4'-hexachlorobiphenyl) and 153, all independent of Ah receptor activation, since these effects were found in Ah-receptor knockouts and Ah low-response mice. No DNA fragmentation was found in either of these preparations with PCB126 (3,3',4,4',5-pentachlorobiphenyl), a coplanar congener.
We have studied the effects of various PCB congeners on viability of acutely dissociated rat thymocytes studied by flow cytometry. We find that ortho-substituted, but not coplanar, PCB congeners cause rapid cell death of thymocytes, and explore various possible mechanisms mediating this cell death.
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MATERIALS AND METHODS |
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An EPICS ELITE ESP flow cytometer was used in these studies. After loading cells with various fluorescent dyes, the thymocytes were excited with an argon laser (488 nm) and 10,000 cells analyzed per sample. On the basis of light scatter, a relatively homogenous population of cells was gated for study. Cells were loaded with appropriate fluorescent dyes for study of viability, calcium concentration, ROS, or mitochondrial membrane potential. Not more than three dyes were used concurrently, and dyes used simultaneously were selected so as to have minimal overlap in emission wavelength.
Cell viability was determined by use of the DNA binding dyes, 7-amino actinomycin-D (7-AAD), or propidium iodide (PI). Either dye was added to cells 35 min before detection at a final concentration of 0.5 µg/ml. Cells were considered to be dead (nonviable) when the fluorescence intensity increased at least ten-fold over that measured in the initial population of living cells. There were no viability assessment differences between 7-AAD and PI other than their absorption spectrum. In some experiments Annexin V-PE was used in combination with 7-AAD in an effort to determine whether cell death was apoptotic or necrotic. In other experiments, we employed fluorescence-conjugated antibodies, FITC-labeled CD4 and PE-labeled CD8 in conjunction with 7-AAD in order to determine whether cell death was selective for subsets of thymocytes.
Intracellular free calcium concentration ([Ca2+]i) was determined using membrane-permeable Fluo-3 AM (100 µM), while ROS level was determined using 5-(and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; 1 µM), as previously described (Boldyrev et al., 1999; Carpenter et al., 1997
). Cells were loaded with both dyes for 1 h in darkness, then resuspended in fresh Tyrodes solution. Mitochondrial membrane potential
m was monitored using 5,5',6,6'-tetraethylbenzimidazolylcardocyanine iodide (JC-1). Cells were loaded with JC-1 (1 µM) for 15 min, and mitochondrial membrane potential was measured as the ratio of orange to green JC-1 fluorescence. Carbonyl cyanide 3-chloro-phenylhydrazone (CCCP) was applied to collapse mitochondrial membrane potential. Cyclosporin A, thapsgargin, rapamycin, and FK 506 were applied to distinguish mitochondrial from endoplasmic reticulum actions.
Single PCB congeners were purchased from Ultra Scientific, and were dissolved in DMSO and diluted such that the final DMSO concentration was not greater than 0.5%. Ionomycin and all fluorescent probes were purchased from Molecular Probes. All other chemicals were purchased from Sigma or Fisher.
Data were acquired in a list mode for off-line analysis with WinList software (Verity Software House, Inc.). All values are reported as mean ± SEM (n = 3 experiments, with at least three trials per experiment). Multiple comparison tests were performed using SAS software (SAS Institute, Inc.).
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RESULTS |
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Loss of plasma membrane integrity (cell viability) after addition of PCB 52 (0.5 µM) was accompanied by an increase of [Ca2+]i as indicated by the Fluo-3 fluorescence (Table 1). The elevation of [Ca2+]i could affect cell viability. In order to test this hypothesis we performed studies in calcium-free Tyrodes solution (no added calcium plus 2 mM EGTA; Fig. 2
). The absence of extracellular calcium did not protect against loss of viability, although it did significantly reduce the PCB-induced elevation of [Ca2+]i, suggesting that most, but not all, of the accumulated calcium comes from calcium entry through the plasma membrane. However even without the substantial rise in the [Ca2+]i, the thymocytes still have significant PCB 52-induced loss of viability. Plasma membrane permeability changes likely are closely associated with the rise in [Ca2+]i since there is good correlation between them after PCB 52 exposure (Fig. 3
). The correlation between the rise in [Ca2+]i and loss of membrane integrity (impermeability to PI) was r2 = 0.925.
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Since the large increase in [Ca2+]i did not appear to be responsible for cell death, additional experiments were designed to further study the effects of ortho-substituted PCBs on mitochondrial and endoplasmic reticulum (ER) functions. As shown in Figure 4, the mitochondrial uncoupler, CCCP, reduced
m and caused an increase in [Ca2+]i. Since calcium uptake by mitochondria depends on the
m, we applied CCCP to block mitochondrial uptake of calcium, then treated thymocytes with PCB 52 in calcium-free medium in order to determine whether there was a resultant calcium release from the ER (Fig. 5
). CCCP alone caused an increase of [Ca2+]i as expected, although it was not as large as the increase caused by PCB 52. Addition of the ortho-substituted PCB in the presence of CCCP caused a further significant [Ca2+]i increase. These results support the conclusion that the ER contributes to the PCB-induced calcium increase. However, there was significantly less of a calcium increase induced by PCB 52 in the presence of CCCP relative to PCB 52 alone. This suggests that the non-planar PCB 52 released calcium from both mitochondria and ER.
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DISCUSSION |
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These observations with thymocytes are very similar to previous reports of the effects of ortho-substituted PCBs on neurons (Carpenter et al., 1997; Kodavanti et al., 1996
; Wong and Pessah, 1996
), in that ortho-substituted, but not coplanar, PCBs caused relatively rapid cell death.
Elevations of [Ca2+]i have been widely viewed as a common final mechanism causing cell death (Siesjo, 1990). While damaging stimuli such as application of ortho-substituted PCBs, triggering mitochondrial depolarization by CCCP, and application of inhibitors of calcium accumulation sites in mitochondria and ER, all increase the [Ca2+]i, this event, by itself, is not the cause of cell death. This was best observed with the studies in Ca-free external media, where the rise in [Ca2+]i upon exposure to ortho-substituted PCBs was substantially reduced, but the degree of cell death was unaltered. This observation clearly shows that while elevation of [Ca2+]i is a component of the injury response, it is not the lone cause of cell death.
Several groups have reported that disregulation of intracellular calcium homeostasis is induced by non-planar PCBs. Nishihara et al.(1985, 1986)
demonstrated that lightly chlorinated non-planar PCBs alter calcium homeostasis by modulation of mitochondrial membrane integrity, while Kodavanti (Kodavanti et al., 1993
; Tilson and Kodavanti, 1997
) reported inhibition of calcium sequestration by mitochondria and ER of neuronal cells induced by ortho-substituted but not coplanar PCBs. Wong et al.(1997)
also invoked a rise in [Ca2+]i as a cause of cytotoxicity. However, they proposed that the calcium came from ryanodine-sensitive stores in the ER. Brown and Ganey (1995)
have shown that non-planar PCBs can induce O2. generation in neutrophils and that this action is dependent on external calcium. Fischer et al.(1996
, 1999)
have shown that ortho-substituted PCBs (but not coplanars) increase [Ca2+]i in beta cells of the pancreas, promoting insulin release, and that the calcium comes from extracellular sources. Carpenter et al.(1997)
reported an elevation of [Ca2+]i induced by ortho-substituted PCBs in cerebellar granule cells. As shown here, a similar elevation of intracellular calcium is induced in thymocytes upon exposure to ortho-substituted PCBs.
Our results support the conclusions of Kodavanti and Pessah and their collaborators concerning the targets of ortho-substituted PCBs. Both mitochondrial and ER calcium regulation are altered by PCB 52, and ryanodine receptors are involved. In addition, we demonstrate that there is damage to the plasma membrane, observed as the increase in both accumulation of calcium from the extracellular medium and a slow leak of PI into the cell. These observations lead us to conclude that the effects of ortho-substituted PCBs are not specific to either mitochondrial or ER membranes, but rather are a general alteration of membrane structure (plasma, mitochondrial, and endoplasmic membranes and probably all other cellular membranes).
ROS also are widely believed to be important mediators of neurotoxicity (LeBel and Bondy, 1991). It has been reported that ortho-substituted PCBs induce superoxide anion (O2.) generation in neutrophils (Ganey et al., 1993
) and that activation of neutrophils is dependent on calcium (Brown and Ganey, 1995
). Oakley et al.(1996)
have shown ROS generation from dihydroxy metabolites of PCBs. Hennig et al.(1999)
have shown that some coplanar congeners damage endothelial cells, and that these actions are potentiated by certain unsaturated fatty acids, presumably via oxidative stress. However, we did not detect any free radical generation by either ortho-substituted or coplanar congeners although the detection of accumulated ROS could have been prevented by the loss of the fluorescent probe (DCF) from the thymocytes with leaky membranes. Even if ROS were generated in thymocytes by PCB 52, the levels would be subsequent to the other observed changes (Table 1
). The generation of ROS in neutrophils and endothelial cells may reflect specific characteristics of certain cell types. In the case of neutrophils, this makes particular sense since they are designed to generate free radicals to defend the host against destruction by pathogens.
An unresolved question is whether the observed death of thymocytes induced by PCB 52 is apoptotic or necrotic. Increases in [Ca2+]i are characteristic of both necrotic and apoptotic cell death in thymocytes (Comment et al., 1992; Orrenius et al., 1989
), as is opening of the mitochondrial permeability channel (Counter et al., 1998
; Lephart, 1996
). One expects necrotic cell death to be accompanied by cell swelling, while apoptotic cell death involves shrinkage. While we observed cell shrinkage, annexin-V labeling did not occur prior to loss of cell impermeability. However, this is not by itself a definitive marker of apoptosis. We did not test for DNA fragmentation. Shin et al.(2000)
and Jeon et al.(2002)
demonstrated ortho-substituted PCB-induced apoptosis in monocytes and spleen cells, respectively, assayed by DNA fragmentation. These studies were performed either on cultured cells (Shin et al., 2000
) or acutely isolated spleen cells incubated for 4 h (Jeon et al., 2002
). In our studies, we rarely investigated cells for more than 1 h. It seems likely that ortho-substituted PCBs induce apoptotic cell death in all types of immune cells, but that the cell death with PCB 52 is too rapid to detect a change in phosphatidylserine location. Our observation that there was no selectivity among thymocyte subsets is consistent with this conclusion.
A decrease in the m is known to be one of the earliest events in apoptosis (Susin et al., 1996
), and this change will induce the opening of the mitochondrial permeability transition channel, which in turn has been hypothesized to lead to the release of mitochondrial apoptosis initiation factors (Marchetti et al., 1996
). When open, this channel causes an abrupt increase of permeability of the mitochondrial inner membrane to solutes and molecules with a mass less than about 1500 (Zoratti and Szabo, 1995
). Our results show that PCB 52 is even more potent in inducing mitochondria depolarization than the classical uncoupler, CCCP. Both PCB 52 and CCCP triggered a concomitant increase in [Ca2+]i. CCCP dissipates mitochondrial membrane potential by shuttling protons across mitochondrial membranes with an acid-dissociable group within the molecule. Ortho-substituted PCBs may not act through this mechanism since they are neutral molecules. The loss of mitochondrial membrane potential cannot be an event subsequent to the elevation of calcium concentration, since membrane potential is reduced prior to measurable elevations of calcium. One possibility is that the mitochondrial membrane potential decrement results from disruption of membrane structure and that cyclosporin A protects cells through mechanisms other than blockade of mitochondrial permeability channel, or that it requires the concurrent loss of mitochondrial membrane potential, rise of [Ca2+]i and loss of membrane integrity to trigger cell death.
The evidence presented here is consistent with the hypothesis originally proposed by Nishihara and colleagues (Nishihara et al., 1985, 1992
) that ortho-substituted PCBs disrupt the structure of lipid membranes. Cyclosporin A, rapamycin, and FK 506 all protect against the cell death, although rapamycin and FK 506 did so only at higher doses. These three agents belong to the immunophilin family of proteins. It is possible that non-planar PCBs act on immunophilins, which may in turn cause immunosuppression, although the evidence to date is insufficient to conclude that this is the mechanism of action.
In summary, we have shown that ortho-substituted, non-dioxin-like PCB congeners kill thymocytes, preceded by a reduction of mitochondrial membrane potential, an elevation of [Ca2+]i, and an increase in the permeability of the plasma membrane. We hypothesize that the PCBs insert into cellular membranes in a relatively non-specific fashion, but that the bulky structure of the ortho-substituted congeners results in a greater disruption of membrane function than the planar, dioxin-like congeners. The actions we describe on thymocytes are similar to those of ortho-substituted PCBs previously reported in several other tissues, and may be responsible for the non-Ah receptor dependent immunosuppression that other laboratories have described.
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ACKNOWLEDGMENTS |
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COI: D.O.C. acknowledges that he has a grant from the NIEHS, and the funding organization does not have control over the resulting publication.
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NOTES |
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