* University at Albany, School of Public Health, Department of Environmental Health and Toxicology, University at Albany, Rensselaer, New York 12144; Wadsworth Center for Laboratories and Research, New York State Department of Health, and School of Public Health, University at Albany, 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 NY 12144
Received November 19, 2003; accepted February 20, 2004
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ABSTRACT |
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Key Words: PCBs; flow cytometry; cerebellar granule cells; intracellular calcium concentration; viability; membrane integrity.
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INTRODUCTION |
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PCBs are a family of 209 chemicals (congeners), each of which consists of two biphenyl rings containing from one to ten chlorine atoms. The properties of individual congeners depend upon both the number of chlorines and their positions around the biphenyl rings. For many years, concerns of PCB toxicity have focused on dioxin-like actions (immunosuppression and carcinogenesis) mediated via activation of the aryl hydrocarbon (Ah) receptor and induction of cytochrome P450 (see Safe, 1994, for review). These effects are primarily due to congeners that have chlorines only in the meta and para positions, which can assume a coplanar, dioxin-like configuration. Congeners with chlorines in the ortho position (closest to the biphenyl bond) are energetically dissuaded from assuming a coplanar configuration.
There is accumulating evidence showing that PCBs are neurotoxic. Studies of children exposed prenatally to PCBs either in two massive poisoning incidents in Asia (Chen et al., 1994; Guo et al., 1994
; Lai et al., 1994
; Yoshimura, 1978
) or U.S. children exposed by maternal consumption of contaminated fish (Jacobson and Jacobson, 1997
; Lonky et al., 1996
) have shown that PCB exposure of the mother is associated with a decrement of cognitive function in the child, which appears to be irreversible. Adults who eat PCB-contaminated fish show decrements in memory, but not in some other nervous system functions (Schantz, et al. 2001
), suggesting a selective toxicity of memory circuits. Similar defects have been observed in monkeys (Rice and Hayward, 1999
; Schantz et al., 1997
) and other species (Tilson and Kodavanti, 1997
).
Laboratory studies have documented toxic actions of PCBs on several cell types, including neurons, and have provided evidence that at least some of these toxicities are mediated by non-coplanar congeners (Brown and Ganey, 1995; Carpenter et al., 1997
; Fischer et al., 1996
; Kodavanti and Tilson, 1997
; Nishihara and Utsumi, 1986
; Seegal et al., 1990
; Wong et al., 1997
). The cellular mechanisms of these actions remain elusive, but there are several hypotheses (see Fischer et al., 1998
, for review). Many of these hypotheses converge on one intracellular messenger, [Ca2+]i. Nishihara and Utsumi (1982
, 1985
) reported that ortho-substituted PCBs alter calcium homeostasis by inducing changes in mitochondrial membrane integrity, while Kodavanti and Tilson (1997)
postulated inhibition of calcium sequestration by intracellular organelles. Brown and Ganey (1995)
have shown that non-coplanar PCBs can induce oxygen radical (O2) generation in neutrophils, and this action is dependent on external calcium. Fischer et al. (1996)
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. Wong et al. (1997)
also suggested that a rise in [Ca2+]i was responsible for cytotoxicity, but proposed that the calcium was released from intracellular ryanodine-sensitive stores in the endoplasmic reticulum.
We performed experiments on cerebellar granule neurons using flow cytometry in an attempt to understand the cellular mechanism(s) of PCB-induced neurotoxicity. Cerebellar granule cells are useful because: (1) 99% of cerebellar cells are granule cells (Zagon, 1977), so it is relatively easy to obtain a large number of similar neurons; (2) they mature in the early postnatal period, so they can be relatively easily dissociated as single cells from the cerebelli of young animals; (3) the granule cells are round and suitable for study by flow cytometry. Our data show that ortho-substituted PCBs can rapidly kill neurons. Cell death is not dependent upon calcium entry from extracellular sources, generation of reactive oxygen species (ROS), or loss of mitochondrial membrane potential.
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MATERIALS AND METHODS |
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Flow cytometry and data analysis:
Using an EPICS ELITE ESP (Coulter) flow cytometer, we preloaded cells with multiple fluorescent probes for simultaneous measurements. All probes used in the experiments are excited by an argon laser (488 nm), and 10,000 cells per sample were analyzed. In order to record from only granule cell neurons, subpopulations of the total cells were selected ("gated") on the basis of forward scatter (which reflects cell size) and side scatter (which reflects cell granularity). With these techniques we could identify a uniform population of cerebellar granule cells and exclude other neurons, as well as distinguish living from dead cells (Carpenter et al., 1997; Dyatlov et al., 1998
). Data was acquired in a listmode for off-line analysis with WinList software (Verity Software House, Inc.). Fluorescence of probes was normalized as percentage of control. The fluorescence of all probes was converted to a linear scale by the following formula provided by the WinList program:
10(decadesex[parameter/resolution])
where decades are the number of decades for the log amplifiers in flow cytometry (4 in this case); parameter is log listmode parameter to convert; and resolution is ADC resolution of the parameter (256 in this case).
Three replicate tubes were used in most experiments, and each data point is the result of at least three experimental replications. All values are reported as mean ± SEM (n = 39), unless specified. Multiple comparison tests were performed using SAS software (SAS Institute Inc.). A p value of less than 0.05 was accepted as being significant.
Determination of membrane integrity and cell viability:
The DNA-binding probes, 7-amino actinomycin-D (7-AAD) (5µg/ml) or propidium iodide (PI) (5µg/ml), were used to determine loss of membrane integrity (Carpenter et al., 1997; Tan et al., 2003
). Either was preincubated for 35 min before adding PCBs. Both PI and 7-AAD are excluded by healthy cells, but enter and bind to DNA when membrane integrity is compromised, which we take to indicate loss of viability. With extended incubations, viable cells will begin to express more fluorescence, likely due to pinocytosis of these probes. The cells were considered to be dead when the fluorescent intensity increased at least ten-fold over the maximal fluorescence in the population of initial healthy living cells ("PI-high", as shown in Fig. 1). Dead cells were excluded from measurements of intracellular calcium concentration, ROS, or mitochondrial membrane potential. As shown in Figure 1, we grouped living cells into two populations: PI-low neurons under the first bar (with maximal fluorescence less than 27) and PI-intermediate neurons under the second bar (with fluorescence between 27 and 294). While the cells under the second bar show greater PI (or 7-AAD) fluorescence than those under first bar, these cells have not become readily permeable and are therefore considered to be viable but damaged. The magnitude of the signal for fluorescence PI or 7-AAD is correlated with membrane integrity (Maftah et al., 1993
). There were no observed differences between 7-AAD and PI other than the emission spectrum. We used PI for all studies except in conjunction with JC-1, where we used 7-AAD to avoid overlap of spectra.
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Detection of mitochondrial membrane potential (m) by JC-1:
Cells were loaded with JC-1 at a final concentration of 1 µM for about 30 min before detection. m was determined as the ratio of orange over green fluorescence, detected separately, or as only orange fluorescence (Reers et al., 1991
; 1995
). When membrane integrity was measured in the same cells as JC-1, it was necessary to use 7-AAD so as to avoid overlap of emission spectra.
Reagents:
PCB congeners (97+% pure) were obtained from Ultra Scientific (North Kingstown, RI). PCBs were dissolved in DMSO and diluted such that the final DMSO concentration was never greater than 0.2%. Control DMSO experiments were always run in parallel to PCB exposures. Ionomycin and all fluorescent probes were purchased from Molecular Probes (Eugene, OR). Dispase II (neutral protease) was ordered from Boehringer Mannheim (GmbH, Mannheim, Germany). All other chemicals were purchased from Sigma Aldrich, Inc. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
Statistical Analysis:
Two-way ANOVA tests were used to determine statistical significance of all results. Results are presented as mean ± SEM. A value of p < 0.05 was considered to be significant.
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RESULTS |
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Figure 2 shows the effects of various PCB congeners, each at a concentration of 10 µM, on membrane integrity (which we assume to be related to cell viability) of cerebellar granule cells (A) and [Ca2+]i (Fluo-3 fluorescence) (B) as a function of exposure time. The ortho-substituted, non-coplanar PCB congeners, PCB 8 (2,4'-dichlorobiphenyl), PCB 28 (2,4,4'-trichlorobiphenyl), PCB 47 (2,4,2',4'-tetrachlorobiphenyl), and PCB 52, all caused loss of membrane integrity of cerebellar granule cells with a potency of PCB 52 > PCB 47 > PCB 8 PCB 28, while three coplanar congeners, PCB 77 (3,4,3',4'-tetrachlorobiphenyl), PCB 80 (3,5,3',5'-tetrachlorobiphenyl), and PCB 81 (3,4,5,4'-tetrachlorobiphenyl), had no effect on cell membrane integrity at the same concentration over this period of time. As shown in Figure 2B, the ortho-substituted PCB congeners also caused an increase in [Ca2+]i, and the relative potency of the different congeners paralleled that for loss of membrane integrity. With the most cytotoxic congeners, there was a decline of Fluo-3 fluorescence with time, presumably reflecting loss of Fluo-3 from the cell because membrane integrity is compromised.
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DISCUSSION |
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Rapid cell death of neurons is usually ascribed to one of four mechanisms: increased toxic level of [Ca2+]i (Berridge et al., 1998; Siesjo, 1990), generation of a toxic amount of ROS (Olanow, 1993
), mitochondrial dysfunction (Cassarino and Bennett, 1999
; Wallace, et al. 1997
), or acute swelling due to entry of sodium and chloride (Rothman, 1992
). The observations reported here indicate, however, that none of these four mechanisms is responsible for the observed PCB-induced cell death.
Two other groups have reported similar cytotoxic actions of ortho-substituted PCBs. Kodavanti and colleagues (1993, 1994
, 1996
, 1997
) have applied somewhat different techniques to cultured cerebellar granule cell neurons and have shown that non-dioxin-like congeners are cytotoxic, potentiate phosphoinositide hydrolysis, and translocation of protein kinase C. These actions are apparent hours after exposure, a considerably longer time than used in our studies, perhaps reflecting differences between acutely dissociated and cultured neurons, and the effects were seen with PCB concentrations in the range of 10 to 100 µM (Kodavanti et al., 1993
). They attributed these actions to altered Ca2+ homeostasis (Shafer et al., 1996
) and suggested that mitochondrial and microsomal calcium transport systems were made dysfunctional. Pessah and colleagues (Wong and Pessah, 1997
; Wong et al., 1997
) have also demonstrated interference with calcium homeostasis by non-dioxin-like congeners, but they suggested the site of action to be the ryanodine receptor in the endoplasmic reticulum in neurons or the sarcoplasmic reticulum of muscle. Our previous investigations using flow cytometry of granule cells have also reported an elevation of [Ca2+]i induced by ortho-substituted congeners (Carpenter et al., 1997
). Our previous and present results are therefore not incompatible with either of these explanations for the elevation of intracellular calcium. However, our results also indicate that the non-coplanar PCB cytotoxicity is not dependent upon the elevation of [Ca2+]i.
Calcium elevation has been widely viewed as a common final mechanism of cell death (Siesjo, 1990). The observation that there was no PCB-induced elevation of intracellular calcium in absence of external calcium provides evidence that the extracellular calcium is the source of the elevation, but this evidence does not rule out the possibility that ortho-substituted PCBs also act by disruption of calcium regulation at intracellular storage sites. However, even if this is the case, PCBs do not appear to kill cells by inducing a massive release of calcium from intracellular stores, since such release should be reflected in a rise of intracellular calcium in calcium-free external medium. Voie et al. (1998)
reported that elevation of [Ca2+]i in human granulocytes by PCB 4 (2,2'-dichlorobiphenyl) was dependent on the extracellular calcium concentration. Cerebellar granule cells from young animals are known to be more tolerant of significant elevations of intracellular calcium than are tolerated by most mature cells, and indeed it has been shown that application of NMDA (Balazs et al., 1988
) or ionomycin (Pearson et al., 1992
), both of which increase intracellular calcium concentration, actually promotes proliferation of granule cells. In addition, a promotion of cell survival by agents that raise calcium has been reported in other types of immature neurons (Collins, 1991
).
While in our analyses the elevation of the [Ca2+]i is not directly responsible for the cell death, this does not mean that the calcium elevation is without physiological importance in either these or other kinds of cells. Fischer et al. (1999) have shown that ortho-substituted PCBs, but not coplanars, cause release of insulin from RINm5F cells, a pancreatic cell line, and that this release is dependent upon entry of extracellular calcium. It appears likely that the mechanism responsible for calcium entry is similar to that described here, since our results clearly demonstrate that ortho-substituted PCBs cause an elevation of [Ca2+]i coming from extracellular sources, and elevation of [Ca2+]i is known to be the trigger for release of insulin.
Like elevations of intracellular calcium, oxygen radicals are widely believed to be common mediators of neurotoxicity (LeBel and Bondy, 1991). Non-coplanar PCBs induce superoxide anion (O2) generation in neutrophils (Ganey et al., 1993
), and this activation of neutrophils is dependent on calcium (Brown and Ganey, 1995
). In addition, Oakley et al (1996)
have shown ROS generation from the 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, using DCF-DA, which is a relatively broad spectrum ROS detector that reacts with most but not all ROS, we did not detect any free radical generation by cerebellar granule cells treated with PCB 52 or PCB 80 (10 µM) in three different media. The generation of ROS in neutrophils and endothelial cells by PCBs may reflect specific characteristics of these cell types. In the case of the neutrophils, this makes particular sense because they are designed to generate free radicals to defend the host from destruction by pathogens.
Ortho-substituted PCBs alter liver mitochondria by inhibition of the electron transport chain and act as an uncoupler (Nishihara and Utsumi, 1987; Nishihara et al., 1986
, 1987
). Thus, one predicted effect of ortho-substituted PCBs would be a reduction of mitochondrial potential. We tested this hypothesis using JC-1 (Reers et al., 1991
, 1995
). Treatment with non-coplanar PCBs, but not dioxin-like PCBs, caused a significant drop in
m, as did the uncoupler, CCCP (2 µM). However, in spite of a dramatic decrement of
m, CCCP caused only a slight decline in cell membrane integrity. Thus, the
m alone cannot be the major factor for the rapid cytotoxicity.
Traditional uncouplers, including CCCP, dissipate m by shuttling protons across mitochondrial membranes with an acid-dissociable group within the molecule (Budd and Nicholls, 1996
). It is not clear how PCBs decrease
m. The mechanism of PCB uncoupling must be different, since PCBs are neutral molecules. LaRocca and Carlson (1979)
have evaluated the relationship between inhibition of Mg-ATPase and the lipophilic property of PCBs and observed a strong negative correlation between PCB-induced inhibition of ATPases and solubility of the PCB congeners. Kodavanti et al. (1996)
have reported inhibition of microsomal and mitochondrial Ca2+ sequestration in granule cells by PCB congeners. The IC50 values for inhibition of microsomal and mitochondrial Ca2+ sequestration were quite similar for both PCB mixtures and single congeners. Since mitochondria and microsomes employ totally different mechanisms for calcium sequestration (uniporter and Ca-ATPase, respectively), one would expect distinct IC50 values if PCBs acted directly on these transport systems. Nishihara and Utsumi (1986)
reported that, when the uniporter was blocked by ruthenium red, administration of PCB 52 still induced calcium release from mitochondria accompanied by mitochondrial swelling. Compromised mitochondrial membrane integrity was further evidenced by the release of endogenous K+ (Nishihara, 1984
) and altered permeability to large molecules such as NADH (Nishihara, 1984
), induced by Kanechlor-400, in which tetrachlorobiphenyls are the major congeners.
Rothman (1985) has described a form of neuronal cell death that results from anionic and cationic influxes induced by high concentrations of the neurotransmitter, glutamate, which causes significant cell swelling leading to cell lysis. The cell swelling and death is rapid and independent of changes in [Ca2+]i. To test whether non-planar PCBs induce cell death via this mechanism, we monitored cell size upon exposure to several congeners (Fig. 8). Coplanar congeners did not alter cell size, but the ortho-substituted congeners caused a time-dependent decrease in cell size. These observations indicate that cell swelling, as a result of NaCl accumulation, is not the mechanism of PCB-induced cell death.
Cell shrinkage is characteristic of apoptotic cell death, not necrotic (Bonfoco et al. 1995), but apoptotic cell death is usually a more delayed response than we see here. Our present data do not allow one to characterize cerebellar granule cell death upon exposure to PCBs as being either necrotic or apoptotic, but the cell shrinkage is more consistent with apoptotic cell death.
Ortho-substituted but not coplanar PCB congeners alter plasma membrane permeability to the relatively large probes, 7-AAD (MW = 1270) and PI (MW = 668), as well as to calcium, of living or injured granule cells. Under normal circumstances these probes are not able to penetrate the impermeable barrier of the plasma membrane. However, after exposure to PCBs there is a degree of accumulation of these compounds, as indicated by the broadening of the peak under "PI-low" cells, and the appearance of injured ("PI-intermediate") cells (Fig. 1), and an increase in [Ca2+]i (Fig. 3).
These results from cerebellar granule cells are very similar to results previously obtained using thymocytes (Tan et al., 2003). Thymocytes are rapidly killed by ortho-substituted PCBs in a dose-dependent fashion. The relative potency of the various congeners is similar to that we describe in this study. The cell death is accompanied by elevations of [Ca2+]i and reduction of
m. There were some differences, in that thymocytes were somewhat more sensitive than granule cells, showing significant cell death at 1 µM concentrations of PCB 52, and there was a significant ROS generation at higher concentrations.
The general similarity of effects of ortho-substituted PCBs on cerebellar granule cell neurons and thymocytes, plus the evidence for disruption of both plasma membrane integrity and intracellular organelles suggests that these compounds may be altering membrane structure. We have examined this possibility using fluorescence polarization dyes. These results are described in the accompanying paper, where evidence is presented that the non-planar PCBs alter the structure and function of several (and likely all) cellular membranes (Tan et al., 2004).
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at School of Public Health, University at Albany, One University Place, B242, Rensselaer, NY 12144. Fax: (518) 525-2665. Email: carpent{at}uamail.albany.edu.
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