Ortho-Substituted PCBs Kill Cells by Altering Membrane Structure

Yuansheng Tan*, Chang-Hwei Chen{dagger}, David Lawrence*,{ddagger} and David O. Carpenter*,§,1

* Department of Environmental Health and Toxicology, {dagger} Department of Biomedical Sciences, School of Public Health, University at Albany, Rensselaer, New York 12144; {ddagger} Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201; and § Institute for Health and the Environment, University at Albany, Rensselaer New York 12144

Received November 19, 2003; accepted February 20, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our previous studies have demonstrated that ortho-substituted PCBs cause a rapid cell death in both thymocytes and cerebellar granule cell neurons, whereas coplanar congeners are without effect at comparable concentrations and exposure times. We have demonstrated that multiple membrane components are altered by these exposures, including the plasma membrane, mitochondria, and endoplasmic reticulum. The present experiments were designed to test the hypothesis that because of their stereochemistry, ortho-substituted congeners cause a greater disruption of membrane integrity than do coplanar congeners, and that this membrane disruption results in altered cellular function and to cell death. To test this hypothesis we have measured fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in thymocytes, cerebellar granule cells, and lipid bilayer vesicles upon exposure to an ortho-substituted PCB congener (PCB 52) and a coplanar congener (PCB 77), and compared results obtained in these studies to those from flow cytometric studies of plasma membrane permeability to large molecules and elevations of intracellular calcium in living cells. The fluorescence polarization of the DPH probe, which inserts into the lipid bilayer, reflects changes in membrane fluidity. In all three preparations we found that whereas fluorescence polarization was unchanged upon exposure to PCB 77, it was reduced significantly by PCB 52, reflecting an increase in membrane fluidity. These observations are consistent with the hypothesis that ortho-substituted PCBs disrupt membrane structure, which alters the function of membrane proteins. In the two cell types we have studied, the disruption is sufficient to cause death of the cell within a brief time.

Key Words: membrane fluidity; fluorescence polarization; lipid bilayer membranes; thymocytes; cerebellar granule cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) are highly lipophilic substances which in animals and humans are stored in body fats, including cellular membranes. Cellular membranes are composed of lipid bilayers into which numerous proteins are inserted which mediate various cellular functions (Steck, 1974Go). In 1972, Singer and Nicolson described the fluid mosaic model of membrane structure, proposing that membrane proteins were effectively a solution of integral proteins floating in a viscous phospholipid bilayer solvent. There have been many demonstrations that substances that alter membrane fluidity alter physiologic function in neurons (Woodson et al., 1976Go) and lymphocytes (Raff and De Petris, 1973Go). Bacteria have been shown to regulate the proportions of saturated and long-chain fatty acids in phospholipids so that they have identical viscosities when grown at different temperatures (Silensky, 1974Go), indicating how important the viscosities of the lipid membrane components are to cellular function.

Although most health effects of PCBs have been ascribed to the dioxin-like, coplanar congeners, there is abundant evidence from cellular and animal studies that the ortho-substituted, nondioxin-like congeners have biological effects in a great variety of organ systems (Hansen, 1999Go). In the nervous system ortho-substituted PCBs have been reported to reduce synthesis of the neurotransmitter dopamine (Seegal et al., 1997Go), block long-term potentiation, an event thought to be related to learning (Hussain et al., 2000Go), cause aberrations in behavior and deficits in memory and learning in rats (Eriksson and Fredriksson, 1996Go; Holene et al., 1998Go), inhibit adenosine triphosphatases (Maier et al., 1994Go), and cause death of cerebellar granule cells (Carpenter et al., 1997Go; Kodavanti et al., 1993Go; Tan et al., 2004Go). Mariussen and Fonnum (2001)Go have reported that ortho-substituted, but not coplanar, congeners inhibit uptake of several different neurotransmitters (dopamine, glutamate, GABA, and serotonin) into brain synaptosomes. Ortho-substituted (but not coplanar) PCBs trigger release of insulin from RINm5F cells (Fischer et al., 1999Go), stimulate generation of superoxide anion in neutrophils (Ganey et al., 1993Go) and stimulate contraction of pregnant rat uterine muscle (Loch-Caruso, 2002Go). PCB 153, a di-ortho persistent congener, causes reproductive failure in mink (Patnode and Curtis, 1994Go), and interferes with sex hormone regulated processes (Bonefeld-Jorgensen et al., 2001Go). Some, but not all, ortho-substituted congeners alter thyroid structure and reduce levels of thyroxine in rats (Ness et al., 1993Go). A statistically significant relationship between PCB 153 levels and free testosterone levels and sperm motility has been found in humans (Richthoff et al., 2003Go).

The mechanism(s) whereby noncoplanar PCB congeners act has been a matter of debate. The thyroid endocrine disruptive effects are probably mediated by binding of the PCBs to either thyroid hormone receptors or binding proteins and to the induction of glutathione-S-transferase in the liver, resulting in more rapid clearing of thyroid hormone from the blood (Brouwer et al., 1998Go; Porterfield, 2000Go). The sex hormone effects are believed to result from agonist actions of the parent PCB or their hydroxylated metabolites at estrogen receptors (Korach et al., 1988Go). The mechanism for reduction of dopamine levels in neurons is believed to be inhibition of the rate-limiting enzyme in dopamine synthesis, tyrosine hydoxylase (Seegal et al., 1997Go). Many of the other actions are associated with elevations of intracellular calcium concentration, either from entry across the plasma membrane or secondary to release from intracellular calcium stores. This has been proposed as the mechanism for the release of insulin from RINm5F cells (Fischer et al., 1999Go) and the contraction of uterine muscle (Bae et al., 1999Go).

Three different hypotheses have been presented to explain at least some of the actions of ortho-substituted PCB congeners. Nishihara and colleagues (Nishihara, 1984Go; Nishihara et al., 1982Go, 1985Go; Nishihara and Utsumi, 1986Go) studied effects of PCBs on isolated mitochondria. They found that ortho-substituted congeners altered calcium homeostasis by inducing changes in mitochondrial membrane integrity, resulting in loss of mitochondrial membrane potential, ATP generation and swelling with increased permeability to large molecules such as NADH and glucose. Kodavanti and colleagues (Kodavanti et al., 1993Go, 1994Go, 1996Go; Shafer et al., 1996Go; Tilson and Kodavanti, 1998Go; Kodavanti and Ward, 1998Go) have studied cerebellar granule cell neurons, where they have demonstrated a variety of effects of ortho-substituted congeners that are not induced by coplanar PCBs. These include inhibition of microsomal and mitochondrial Ca2+ sequestration, effects on phosphoinositide hydrolysis, protein kinase C activity, and cytotoxicity leading to cell death. More recently they have demonstrated that lower chlorinated, di-ortho-substituted congeners inhibit nitric oxide synthases (Sharma and Kodavanti, 2002Go). The central hypothesis from these investigators is that ortho-substituted PCBs alter calcium homeostasis and intracellular second messengers in both mitochondria and endoplasmic reticulum. Pessah and collaborators (Wong and Pessah, 1996Go, 1997Go; Wong et al., 1997Go, 2001Go) have also focused on the effects of ortho-substituted PCBs on calcium regulation, but have presented evidence from skeletal and cardiac muscle as well as from neurons that a critical site of action is the ryanodine receptor, which controls a calcium release channel in the endoplasmic reticulum. Their studies have been primarily with PCB 95, which activates ryanodine receptors via an immunophilin-dependent mechanism, leading to a depletion of the endoplasmic reticulum calcium store. The central hypothesis from this group is that the ryanodine receptor is the major target of ortho-substituted congeners.

Our studies on acutely dissociated cerebellar granule cells (Carpenter et al., 1997Go; Tan et al., 2004Go) and thymocytes (Tan et al., 2003Go) using flow cytometry have demonstrated that ortho-substituted PCB congeners cause a rapid cell death in both cell types. While the PCB action is accompanied by elevations of intracellular calcium, our evidence indicates that the rise in intracellular calcium is not the cause of the cell death. We also have evidence implicating both mitochondrial and endoplasmic reticulum changes in responses of both cerebellar granule cell neurons and thymocytes to ortho-substituted PCBs. The present studies were designed to test the hypothesis that these congeners exert a relatively nonspecific alteration of all cell membranes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All methods for preparation of cerebellar granule cell neurons for flow cytometry are presented in the accompanying article (Tan et al., 2004Go), while those for preparation of thymocytes are presented in a previous publication (Tan et al., 2003Go). In brief, the cerebellum or thymus gland was removed from 10–15 day-old Sprague-Dawley rat pups, and cut in slices of approximately 500 µm. Thymus pieces were ground between two frosted glass slides, while brain slices were enzymatically dissociated to obtain single cells. The single cells were filtered and placed in fresh Tyrode's solution. Aliquots of 1 x 106 cells were then incubated with appropriate fluorescent dyes as described in detail in the above articles. Cell viability was monitored by use of the DNA binding dyes, propidium iodide (PI), or 7-amino actinomycin-D (7-AAD). These dyes were also used to monitor plasma membrane injury, where some increase in labeling indicates sublethal changes in membrane permeability. The fluorescence values are reported normalized to the control values, taken as 100%. Intracellular calcium concentration was monitored by use of Fluo-3 AM.

A Perkin-Elmer MPF fluorescence spectrophotometer was used to measure steady-state fluorescence polarizations, following the protocol of Lasner et al. (1995)Go. The fluorescence probe, 1,6-diphenyl-1,4,5-hexatriene (DPH), was incorporated into the hydrocarbon chains of the cell membrane lipids (Pap et al., 1994Go) as described by Wang and Chen (1993)Go and Chen et al. (1998)Go. By monitoring the changes in DPH fluorescence polarization, one can investigate the effects of lipophilic agents on membrane fluidity. DPH was initially prepared as a stock solution in tetrahydrofuran at concentrations between 10–7 and 10–5 M. A small amount of the DPH solution was then added to thymocytes or cerebellar granule cell neurons. DPH-containing thymocytes or cerebellar granule cell neurons were incubated for 30–60 min at room temperature before the addition of an appropriate concentration of the PCB. Steady-state fluorescence polarization was monitored in the absence and presence of PCBs at a time and concentration showing a membrane integrity change and calcium increase, but no obvious cell death.

The effects of PCBs on lipid bilayer vesicles prepared from synthetic L-3-dipalmitoyl phosphatidyl choline (DPPC) were also investigated. The procedures for preparing lipid bilayer vesicles from synthetic DPPC have been described previously (Chen et al., 1996Go). In brief, DPPC (5 mg in 2 ml distilled water) in a flask filled with nitrogen gas was sonicated, using a sonicator (Brason 2210) with a power output of 50W, at 5°C above the transition temperature of DPPC (42°C) for about 30 min. A small amount of DPH solution was then added to the lipid bilayer vesicles. DPH-containing lipid bilayer vesicles with a molar ratio of DPPH to lipid of 1/500 were then incubated for 30–60 min at room temperature, before adding an appropriate concentration of one or the other PCB.

Steady-state fluorescence polarization (P) was measured at room temperature, where the excitation wavelength was at 360 nm and the emission wavelength at 430 nm. The value of P was calculated according to P = (Ivv – gIvh)/(Ivv + gIvh), where g is a grating correction factor which is equal to Ihv/Ihh, I is the intensity of the light, v and h denote the vertical and horizontal orientation of the polarizers, and the first and the second subscripts refer to the excitation and emission light, respectively.

The statistical tests used varied with the experiment. Two-way ANOVA was used in experiments such as those shown in Figure 1. A one-way ANOVA (linear regression) was performed on the data in Figure 2, and multiple comparisons tests were applied to the bar graph figures. The Student's t-test was performed for the simple paired tests, with the level p < 0.05 considered to be significant.



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FIG. 1. Time-dependent accumulation of PI in granule cell neurons exposed to various PCB congeners (10 µM). Only those cells under the bar labeled "PI-low cells" (Figure 1 of Tan et al., 2004Go) were gated. PI fluorescence was normalized to 100% of control in each group. *Significantly different from control (p < 0.05 from two-way ANOVA). All data show mean ± SEM.

 


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FIG. 2. The time-dependent accumulation of PI in PI-low granule cell neurons upon exposure to PCB 52 is accompanied by an accumulation of calcium, as indicated by Fluo-3 fluorescence. As shown in the previous article (Tan et al., 2004Go), Fluo-3 fluorescence decreases when the cell membrane integrity is compromised after exposure to PCB 52. Therefore the data were truncated at the point where the Fluo-3 decrement began to occur. Data were pooled from the experiments in Figure 1 of the article by Tan et al. (2004)Go by gating on only those cells with PI fluorescence values (channel numbers) in the range of 20 to 55. r2 = 0.86, p < 0.0001.

 

    RESULTS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Figure 1 shows the actions of various PCB congeners on the accumulation of PI in living granule cells. When the cell loses membrane integrity completely, the PI fluorescence increases by about four orders of magnitude (see Figure 1 in Tan et al., 2004Go). In this experiment we gated only on those neurons under the bar labeled "PI-low" in Figure 1 of Tan et al. (2004)Go. These are cells in which membrane integrity is intact and PI is mostly excluded. The results show that the ortho-substituted PCBs (PCB 52 < PCB 47 < PCB 8 < PCB 28) caused a small, time-dependent but significant PI accumulation ("PI-intermediate"), whereas the coplanar congeners (PCBs 77, 80, and 81) had no effect different from the control and DMSO control. We interpret this to indicate a slow but increasing loss of plasma membrane integrity, which allows a slow leak of PI (MW = 668) into the cell. These observations indicate that the ortho-substituted PCB congeners alter plasma membrane permeability to PI with relative toxicity closely matching that found for induction of cell death (Tan et al., 2004Go).

Figure 2 is a plot of Fluo-3 fluorescence against the PI fluorescence in these cells that are presumed to be injured. The slow accumulation of small amounts of PI in the cells is accompanied by an increase in intracellular calcium concentration, as reflected by Fluo-3 fluorescence. Thus these observations provide evidence that ortho-substituted PCB congeners alter plasma membrane permeability.

Figures 3 and 4 show the effects of exposure to two PCB congeners of equal molecular weight on steady-state DPH fluorescence polarization in cerebellar granule cells and thymocytes, respectively. The PCBs were at a concentration of 2 µM in the granule cells, and at 0.5 µM in the thymocytes. These concentrations were chosen because they are about the threshold concentrations of PCB 52 for clear effects on the accumulation of DNA binding dyes and elevations in intracellular calcium in these two cell types after a 15-min exposure. In both cell types the fluorescence polarization decreased significantly upon incubation with PCB 52, whereas it was unchanged in presence of a similar concentration of the coplanar congener, PCB 77.



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FIG. 3. Effects of the coplanar PCB 77 and the ortho-substituted PCB 52 on steady-state fluorescence polarization of DPH in thymocytes. The cells were incubated with the PCB congeners (0.5 µM) for about 15 min before test. *Significantly different from control, p < 0.01, by multiple comparison test.

 


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FIG. 4. Effects of the coplanar PCB 77 and the ortho-substituted PCB 52 on steady-state fluorescence polarization in cerebellar granule cell neurons. The neurons were incubated with the PCB congeners (2 µM) for about 15 min before measurements. *Significantly different from control, p < 0.01, by multiple comparison test.

 
Figure 5 shows a similar experiment on DPPC lipid bilayer vesicles. After a 10-min incubation with PCB 52 the fluorescence polarization declined by almost 50%, whereas a similar concentration and duration exposure to PCB 77 had no significant effect.



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FIG. 5. Effects of the coplanar PCB 77 and the ortho-substituted PCB 52 on steady-state fluorescence polarization in artificial lipid vesicles. Lipid vesicles were incubated with PCB congeners (0.5 µM) for about 10 min before test.*Significantly different from control, p < 0.01, by multiple comparison test.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
These observations provide support for the hypothesis that ortho-substituted PCBs disrupt membrane structure in a relatively nonspecific fashion, whereas there is little or no apparent effect of the coplanar congeners. We believe this effect to be a general alteration of membranes, and not effects on only certain cells or certain membranes. The reason for this conclusion is that our previous studies and others have demonstrated effects on mitochondria, endoplasmic reticulum, and plasma membranes and in a variety of cell types. Furthermore, lipid bilayer vesicles show the same direction of alteration of fluorescence polarization by PCBs as is seen in the cells.

There is precedent for this hypothesis. As discussed above, Nishihara and colleagues proposed disruption of mitochondrial membranes by ortho-substituted PCBs as long ago as 1982. Lopez-Aparicio et al. (1997)Go have reported on studies on rat renal tubular cell cultures exposed to PCBs. They found that Aroclor 1248 caused a dose-dependent increase in membrane fluidity. They also studied two PCB congeners, and found that an increase in fluidity was induced by PCB 153, but not by PCB 77. These observations are consistent with our hypothesis. It is interesting that drugs such as cyclosporin A, which rapidly partitions into membranes, is known to cause significant changes in membrane structure, including a change in lipid domain morphology (Soderlund et al., 1999Go). Cyclosporin A, which is known to block the mitochondrial permeability transition channel, was found to be the most effective of all agents tested in preventing loss of cell viability in thymocytes (Tan et al., 2003Go).

Disruption of the membrane structure can cause changes in ion permeabilities through voltage- or ligand-gated channels, as well as changes in activity of membrane bound enzymes. Disruption of membrane structure can occur at any membrane, whether the plasma membrane or membranes of intracellular organelles such as mitochondria or endoplasmic reticulum, and if such disruption of structure occurs one would expect altered function. We are certainly not proposing that all effects of ortho-substituted congeners are necessarily mediated by this mechanism, but it is likely that some of those involving elevations in intracellular calcium and activation or inhibition of membrane-bound enzymes and second messengers may be.

Our studies of fluorescence steady-state polarization have been done with two PCB congeners of the same molecular weight and the same number of chlorines, but with the chlorines at different positions around the biphenyl ring. Because the chlorine atoms are relatively bulky, when there are chlorines in the ortho positions close to the biphenyl bond on each side they cause the angle between the biphenyl rings to increase, whereas with the chlorines in the meta and para positions, the molecule assumes a planar configuration. There is no reason to assume that the ortho-substituted and coplanar congeners differ greatly in their ability to dissolve in biological membranes, but our observations suggest that the bulky, three dimensional structure of the ortho-substituted congeners causes sufficient perturbation of the membrane lipids so as to cause changes in physiologic function. The physiologic changes that then occur will obviously be dependent upon the specific proteins in different cell membranes. These will be different in neurons as compared to thymocytes, and different yet in other types of cells.

Our results suggest that low or submicromolar concentrations of some ortho-substituted PCBs alter membrane structure, and indicate that this is accompanied by an increase in membrane fluidity as well as accompanying disruption of plasma, mitochondrial and endoplasmic reticulum membrane functions. This may be the mechanism whereby ortho-substituted PCBs alter the function of a variety of cells. The fact that not all cell types are affected similarly or at the same concentration may reflect differences in lipid composition and membrane proteins.


    NOTES
 

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. E-mail: carpent{at}uamail.albany.edu.


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