Interactive effects of environmentally relevant polychlorinated biphenyls and dioxins on [3H]phorbol ester binding in rat cerebellar granule cells.

Polychlorinated biphenyls (PCBs) are persistent contaminants that exist as complex mixtures in the environment. One problem faced by risk assessors is that the possible interactive effects of specific PCB congeners and related chemicals found in environmental and biological samples have not been systematically investigated. Some PCBs perturb Ca2+ homeostasis and cause protein kinase C (PKC) translocation in neuronal cell cultures and in brain homogenate preparations at concentrations where no cytotoxicity is observed, and these systems are necessary for the growth and normal functioning of neurons. The changes in second messenger systems appear to be associated with the extent of noncoplanarity of the PCB molecule. We studied the interactive effects of selected PCB congeners, a PCB metabolite, and a dioxin on PKC translocation, as determined by [3H]phorbol ester binding in cerebellar granule cells. The binary combinations included coplanar and noncoplanar PCB congeners or PCB congeners with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/PCB metabolite. In addition, we tested the interactive effects of several PCB congeners (three or more) found in environmental samples such as human milk and blood, contaminated fish, and brain samples from PCB-treated animals. The results indicated that 1) the coplanar congener [3,3',4, 4'-tetrachlorobiphenyl (TeCB)] did not alter the in vitro activity of the noncoplanar (2,2',5,5'-TeCB) or coplanar [4, 4'-dichlorobiphenyl (DCB)] congeners; 2) binary mixtures of active PCB congeners (2,2',4,4'-TeCB and 2,2'-DCB; 2,2'-DCB and 3,5-DCB; 2,2',3,5',6-PeCB and 2,2',4,4',5-PeCB) interact in a dose-additive manner; 3) TCDD did not alter the activity of either coplanar (3,3', 4,4'-TeCB) or noncoplanar (2,2',5,5'-TeCB) congeners; 4) the interaction between the parent PCB congener and hydroxy metabolite of PCB is additive; 5) PCB congener mixtures at the ratios found in environmental samples are biologically active; and 6) there was no indication of synergism in any of the combinations studied. These results suggest that the biological effects of binary mixtures of PCB congeners fit a dose-additive model, indicating that there is a specific site of action for these PCB congeners which is independent of the aryl hydrocarbon receptor. Environmental mixtures contain mostly noncoplanar PCB congeners, and because they appear to be biologically active, the potential human health risk by this group of chemicals should be considered in the risk assessment of PCBs.

Polychlorinated biphenyls (PCBs) are persistent coninants that exist as complex mixures in the environment. One problem faced by risk assessors is that the possible interactive effects of specific PCB congeners and related chemicals found in environmental and biological samples have not been systematically investgated. Some PCBs perturb Ca2+ homeostasis and cause protein kinase C (PKC) translocation in neuronal cell cultures and in brain homogenate preparations at concentrations where no cytotoxicity is observed, and these systems are necesary for the growth and normal finctioning of neurons. The changes in second messenger systems appear to be associated with the extent of noncoplanarity of the PCB molecule. We studied the interactive effects of selected PCB congeners, a PCB metabolite, and a dioxin on PKC translocation, as determined by [3HIphorbol ester binding in cerebellar granule cells. The binary combinations included coplanar and noncoplanar PCB congeners or PCB congeners with 2,3,7,8-tetrachlorodibenzo-p-doin (TCDD)/PCB metabolite. In addition, we tested the interactive effects of several PCB congeners (thee or more) found in environmental samples such as human milk and blood, contaminated fish, and brain samples from PCB-treated animals. The results indicated that 1) the coplanar congener [3,3',4,4'-tetrachlorobiphenyl (TeCB)] did not alter the in vitro activity of the noncoplanar (2,2',5,5'-TeCB) or coplanar [4,4'-dichlorobiphenyl (DCB)] congeners; 2) binary mixtures of active PCB congeners (2,2',4,4'-TeCB and 2,2'-DCB; 2,2'-DCB and 3,5-DCB; 2,2',3,5',6-PeCB and 2,2',4,4',5-PeCB) interact in a dose-additive manner; 3) TCDD did not alter the activity of either coplanar (3,3',4,4'-TeCB) or noncoplanar (2,2',5,5'-TeCB) congeners; 4) the interaction between the parent PCB congener and hydroxy metabolite of PCB is additive; 5) PCB congener mi at the ratios found in environmental samples are biologically active; and 6) there was no indication of synergism in any of the combinations studied. These results sugst that the biological effet of binary mixtures of PCB congeners fit a dose-additive model, indicating that there is a specific site of action for these PCB congeners which is independent of the aryl hydrocarbon receptor. Environmental mis contain mosdy noncoplanar PCB congeners, and because they appear to be biologically active, the potential human health rik by this group of chemicals should be considered in the risk assessment of PCBs. Key (24). A significant perturbation in Ca2+ homeostasis and PKC can trigger processes that could alter the functioning and growth of the neurons (25) and ultimately could lead to cytotoxicity (26). Hence, it is possible that PCB-induced alterations in Ca2+ homeostasis and PKC may form a biochemical basis for the observed neurological effects. Human exposure to PCBs occurs mainly via oral ingestion of contaminated dairy products, meat, and fatty fish, all of which contain a mixture of noncoplanar and coplanar congeners (5,27). The degree to which the constituents of these mixtures may interact by synergism, additivity, or antagonism of effects has not been extensively studied (28)(29)(30)(31)(32)(33). Most research has focused on commercial mixtures or a few congeners. The present study was undertaken to understand the interactive effects of several PCB congeners and dioxins that have different degrees of potency on PKC translocation in neuronal preparations. In the neuron, PKC activation/translocation could be due to increased intracellular free Ca2+, formation of diacylglycerol, and increased levels of free fatty acids such as arachidonic acid and lysophospholipids (34). Sustained activation of PKC has been reported to be involved in Polyhalogenated aromatic hydrocarbons such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins ( Fig. 1) (1-3) are persistent environmental pollutants and bioaccumulate throughout the food chain, thus posing a potential health risk to humans and animals (4,5). Neurotoxicity has been reported following environmental and experimental exposure to PCBs (6-9). These effects indude memory deficits and intellectual dysfunction in children born to mothers who ate PCB-contaminated food (10)(11)(12)(13)(14). In utero exposure to PCBs in animals causes alterations in locomotor activity (15,16) and affects cognitive function (7,17). The mechanism(s) by which PCBs cause these neurological effects remains to be elucidated.
Animals. Timed pregnant female (gestational day 16) Long-Evans hooded rats were obtained from Charles River Laboratory (Raleigh, NC) and housed individually in American Association for Accreditation of Laboratory Animal Care (AAALAC)-approved animal facilities. Food and water were provided ad libitum. Temperature was maintained at 21 ± 2'C and relative humidity at 50 ± 10% with a 12-hr light/dark cycle (0700-1900 hr). Cerebellar granule cell culture. We isolated granule cells from rat (6-to 8-day-old pups) cerebella by the enzymatic disruption of cells as outlined by Gallo et al. (39) with modifications (18). The final cell suspension contained 1.0 x 106 cells/ml, and 1.5ml cell suspension was added into each well of 12-well culture plates, precoated with poly-L-lysine. These culture plates were maintained in an incubator at 37°C under 5% CO2/95% filtered room air. Cytosine arabinoside (5 pM) was added after 24-48 hr of plating to prevent the growth of nonneuronal cells. We routinely examined the cultures by immunocytochemistry for neuron-specific enolase (neurons) and glial fibrillary acidic protein (glial). The estimated purity of neurons always exceeded 95%. f3H]Phorbol ester binding in cerebellar granule cells. Cerebellar granule cells grown on 12-well culture plates (Costar no. 3512, optically dear, virgin polystyrene and radiation sterilized, Cambridge, Massachusetts) were tested at 7 days in culture for [3H]phorbol ester binding according to the method oudined by Vaccarino et al. (40). Briefly, the monolayers were washed with Locke's buffer (NaCl, 154 mM; KCl, 5.6 mM; NaHCO3, 3.6 mM; CaCl2, 2.3 mM; D-glucose, 5.6 mM; HEPES, 5 mM; pH 7.4) containing 0.1% fatty acid-free bovine serum albumin.   Concentrations .50 pM, for most of the PCB congeners, were not completely soluble in the assay buffer. An equal amount of DMSO was added to controls. After incubation, the medium was aspirated, and cells were washed three times with Locke's buffer and suspended with 1 ml of 0.1 M NaOH. An aliquot of this sample (0.7 ml) was added to 9 ml Ultima Gold (Packard, Meriden, CT), and the radioactivity was determined using scintillation spectroscopy. A small aliquot was used for protein determination (41). We determined nonspecific binding in the presence of 1.6 pM phorbol myristate acetate, which was always <20% and subtracted from all the values. The unit of [3H]PDBu binding was femtomoles per milligram protein per 15 min.
Interactive effects of environmental mixtures on PH]PDBu binding. Biota in the environment (including the human population) are exposed to mixtures of PCB Volume 106, Number 8, August 1998 * Environmental Health Perspectives congeners, and all environmental samples contain several of these congeners. In the present study, mixtures selected for testing included the ones found in human milk (mixtures 1 and 2), contaminated lake fish (mixture 3), blood of general population (mixture 4), and in brains of pups born to rats exposed to commercial PCB mixtures (mixture 5). Artificial mixtures were generated to reflect the ratio of the major constituent PCB congeners in these environmental mixtures (Table 1). Figure 10 shows the effects of the selected environmental mixtures on [3H]PDBu binding in rat cerebellar granule cells. All the mixtures increased [3H]PDBu binding in a concentration-dependent manner (Fig.  10). Post hoc analysis indicated that mixtures 1, 4, and 5 were significantly different from control starting at 30 pM, whereas mixtures 2 and 3 were significantly different from control starting at 50 pM (Fig. 10). Of all the selected mixtures, mixture 3 was the most active. To understand the interactions between PCB congeners in these mixtures, the data were fit into a previously established "logistic second-order response surface model," which is based on dose-addition assumption [for details, see Svendsgaard et al. (45]. The observed and predicted E50 values (the concentration that increases the control activity by 50%) along with their 95% confidence limits (CL) are presented in Table 2

Discussion
In the environment, humans are exposed to complex mixtures containing many PCB congeners and contaminants such as dioxins and furans. Because of different metabolic contaminants, the pattern of composition in biological and environmental samples differ from that of the original commercial mixture (5,46). Human populations are exposed to complex mixtures of PCBs, but most laboratory research has focused on commercial mixtures or individual congeners. The objective of this research was to understand more completely the potential interactions of some of the components in these mixtures. [3H]PDBu, [3H]phorbol 12,13-dibutyrate. The binding was represented as percentage of control (361 ± 12 fmol/mg protein/15 min; n = 29). Values are means ± standard errors of five to six preparations, assayed in triplicate.
have been restricted to nonneuronal systems. For example, there are few reports indicating that the effects of these compounds are additive using end points of toxicokinetics (29,30), liver enzyme induction (47,48), teratogenicity (28), or immunotoxicity (49). An in vivo study by Sargent et al. (50), however, indicated more-than-additive effects of 2,2',5,5'-TeCB and 3,3',4,4'-TeCB on hepatocytes and lymphocytes. On the other hand, the immunosuppressive activity of PCB mixtures studied by Harper et al. (51) was found to be less than additive. These investigators conduded that the TEF (toxic equivalency factor) approach may overestimate the toxicity of these compounds. Jansen et al. (52) reported that 2,2',5,5'-TeCB was estrogenic and 3,3',4,4'-TeCB was antiestrogenic, suggesting these compounds may be antagonistic in combination.  (54) found that the toxic potency of mixtures of PCBs, furans, and dioxins was additive in causing early life-stage mortality in lake trout. Recently, van Birgelen et al. (32) reported synergistic effects of 2,2',4,4',5,5'-HCB and TCDD on hepatic porphyrin levels in the rat. These reports suggest that there are multiple mechanisms for the effects of PCBs and related compounds. Although there are several reports on the effects of PCB congeners in neural tissue (6,7,15,17,(55)(56)(57), there are no reports on the potential interactive effects of these congeners in the nervous system. Our previous structure-activity relationship studies (20,22) used stimulation of [3H]PDBu binding as an indicator of PKC translocation due to altered intracellular Ca2+ homeostasis by PCBs. Both Ca2+ homeostasis and PKC translocation, which are involved in the neurotoxicity of a variety of environmental chemicals (26,37,58), were affected by PCB congeners that have a noncoplanar configuration (18,19,59). Higher concentrations and longer exposures to 2,2'-DCB, a noncoplanar PCB, result in cytotoxicity (18). The present study extends our structure-activity relationship work by examining binary combinations of PCBs, a hydroxy-PCB or TCDD to determine 1) if the active congeners are "dose additive," indicating that they act through a common site, or "effect additive," indicating that, although the end point ([3H]PDBu binding) is common, the congeners act through independent sites (60); 2) if the nonactive congeners enter the active site and are ineffective at inducing [3H]PDBu binding or if these congeners simply are unable to enter the active site; and 3) if a dioxin or a hydroxy-PCB alters the response of PCBs. In addition, mixtures were generated to match the ratio of congeners found in environmental and biological samples and studied their effects on [3H]PDBu binding in neuronal cultures.
The inactivity of 2,2',6,6'-TeCB and 3,3',4,4'-TeCB could be due either to their inability to enter the active site or to inability to elicit the response after entering. When these congeners were tested in combination with active congeners, there was no significant interaction. The nonactive coplanar congener (3,3',4,4'-TeCB) also did not alter the activity of either the noncoplanar (2,2',5,5'-TeCB) or active coplanar (3,5-DCB) congeners. The inactive, noncoplanar congener 2,2',6,6'-TeCB also failed to alter the effects of an active PCB congener (2,2',4,. When the dose response of 3,3',4,4'-TeCB was measured with 2,2',6,6'-TeCB, no interaction was seen. The nonactive congener failed to block the effects of the active congeners, suggesting that these nonactive congeners do not bind to the response-inducing site. E50, effective concentration that increases the control activity by 50%. "Observed E. values (mean and 95% confidence limits) were calculated from the regression line fit to the linear portion of the curve. bPredicted Em values (mean and 95% confidence limits) were obtained from the logistic second order response surface model based on dose-addition assumption described in Svendsgaard et al. (45).
Cindicates that the mixture increased 13H]phorbol ester binding significantly, but the increase was below E5m at 100 pM. A pair of active congeners, 2,2'-DCB with 2,2',4,4'-TeCB (Fig. 5), in which one congener was more active than the other, was tested (maximum stimulations of 163% and 93% for 2,2'-DCB and 2,2',4,4'-TeCB, respectively). If the congeners with different activities acted at different sites to increase [3H]PDBu binding, the results would be additive as in an "effect additive" model. If, on the other hand, the congeners act at the same site, the effects would not be additive, fitting a dose additive model. As shown in Figure 5, the interactions between 2,2'-DCB and 2,2',4,4'-TeCB indicate a less-than-additive effect, but no statistically significant interaction was found. This interaction is not limited due to saturation of effect (end point measured is [3H]PDBu binding) because we have previously reported that [3H]PDBu binding can be increased as high as 3.5-fold (19). Solubility of PCB congeners might play a key role at concentrations greater than 100 pM.
Following these experiments, a second pair of congeners was selected with greater difference in activity between two congeners where an interaction would be more clearly demonstrated. The results of the binary combinations of a very active congener, 2,2',3,5',6-PeCB (maximum stimulation of 150%), and a less active congener, 2,2 ',4,4',5-PeCB (maximum stimulation of 56%), are shown in Figure 6. This combination resulted in a significant interaction (Fig. 6), where the less active congener, instead of adding to the total [3H]PDBu binding, decreased this effect, indicating that the less active congener interacted at the same site but lowered the total activity, thus resulting in a less-thanadditive response. On the other hand, the response of a mixture of 3,5-DCB with and without 2,2'-DCB (which have similar activity) showed simple additive response and no interaction. A similar additive interaction was observed with the parent PCB and the hydroxy metabolite (Fig. 9). Taken together, these data indicate that the increase in [3H]PDBu binding induced by certain PCB congeners and their hydroxy metabolites results from interaction at a specific site and not a generalized nonspecific interaction. In addition, the nonactive congeners did not interfere with the active congeners, indicating that they do not enter the active site.
The results of the present study confirm and extend the hypothesis that the PCB congeners in neuronal system do not act through a dioxinlike mechanism. For instance, TCDD at concentrations up to 200 nM was ineffective in this system (data not shown). In addition, TCDD (100 nM) did not interact with an active congener (2,2',5,5'-TeCB) or a nonactive congener (3,3',4,4'-TeCB). These results indicate that PKC translocation in neuronal cultures represented as [3H]PDBu binding is independent of the aryl hydrocarbon receptor mode of action and that the current TEF approach does not include effects reported in the present study. Therefore, application of the TEF approach based on TCDD-like activity for risk assessment of PCBs would be inappropriate and must be used with caution.
Complex mixtures were generated to more nearly mimic human exposure. These mixtures are representative of the ratios of the main constituents of the PCB load, but no attempt was made to match a total dose. Mixtures 1 and 2 were generated to represent the ratios of PCB congeners found in human milk (27,38) and would be a measure of infant exposure. Mixture 3 represents the PCB congener ratio found in Wisconsin fish (61), which represents adult and infant exposures. Mixture 4 is the congener's mix from human blood representing maternal exposure (6Z. Mixture 5 represents the ratio of PCB congeners detected in the brains of rats at postnatal day 21 that had been exposed to PCBs from conception to weaning via feeding dams with adulterated chow containing the commercial mixture Aroclor 1254 (55). All these mixtures increased [3H]PDBu binding. Mixture 3 was the most active among the mixtures tested, followed by mixtures 4 and 5. To understand the interactions between congeners in these mixtures, the data were fit in a previously established empirical model based on dose additive assumption. The observed E50 values are dose to the predicted values obtained from the empirical model, suggesting that the interactions between PCB congeners in these mixtures may follow dose additivity.
In summary, the present data suggest that the biological effects of some PCB congeners are dose additive and that the coplanar congeners do not alter the activity of noncoplanar congeners, indicating a common mechanism for all noncoplanar congeners distinct from that of coplanar congeners. Some interactions showed inhibition of a very active congener by a moderately active congener, indicating that the congeners act at the same site. The dose-additive model, which implies a single site of action, seems to be a more appropriate model for predicting the results. There was no indication of synergism in any of the combinations studied. Because samples from human and environmental mixtures of PCB congeners were biologically active, careful attention must be paid for the role of noncoplanar congeners in PCB exposure assessment process. N t Excellence in basic research at the National Institute of Environmental Health Sciences NIEHS scientists and grantees perform basic studies of our susceptibility to environment-related disease: Demonstrating that a carcinogen in cigarette smoke (benzo(a)pyrene) alters part of a gene to cause lung cancer...showing the effects of fetal exposure to PCBs...developing a strain of mouse that lacks functional estrogen receptors and that helps evaluate how some pesticides and other estrogen-like compounds might affect development and reproduction...discovering the genes for breast, ovarian, and prostate cancers...finding women's.,optimal days of fertility...seeking to reverse the damage from lead exposure...finding alternatives to tradit pinpointing the functions of specific genes by eliminating them from specially bred mouse Ith using ordinary yeast cells, to isolate and clone genes and other fragments of genetic materi the effects of urban air on lung function...