Congener-specific polychlorinated biphenyl-induced cell death in human kidney cells in vitro: potential role of caspase

Polychlorinated biphenyls (PCBs) are among the most widespread and persistent pollutants in the global environment. Coplanar and noncoplanar PCBs have been shown to cause congener-specific apoptosis mediated neurotoxicity in rats. Very few, if any, such studies have been reported on human renal cell toxicity. The authors report here caspase-dependent or caspase-independent renal toxicity, as measured by apoptotic death induced by PCBs, depending on the planarity of congeners PCB-77 (coplanar) and PCB-153 (noncoplanar) in human kidney cells (HK2) in vitro. The authors have combined morphological and biological techniques to discover the relevance of apoptosis in renal proximal tubule cell death induced by these two PCB congeners. Treatment with both PCB congeners caused accelerated apoptosis in a time- and concentration-dependent manner. Based on our findings using human kidney (HK2) cells, there was more apoptosis-mediated loss of cell viability by non-ortho-substituted PCB-77 when compared to PCB-153. A significant increase of caspase-3 expression through immunoblot studies showed the involvement of apoptosis by PCB-77 compared to none by PCB-153. The broad-spectrum caspase inhibitor z-VAD-fmk showed increased cell death when treated by PCB-153, but not by PCB-77, confirming that caspase inhibitor induced a switch in the mode of cell death. It is reasonable to assume that apoptotic cell death in the renal proximal tubule cells treated by PCBs may have both caspase-dependent and caspase-independent pathways.     

Inhibition of gap junctional intercellular communication by noncoplanar polychlorinated biphenyls: inhibitory potencies and screening for potential mode(s) of action.

Polychlorinated biphenyls (PCBs), a structurally diverse group of environmental pollutants, are effective promoters in two-stage cancer models, which implies that epigenetic mechanisms are involved. Inhibition of gap junctional intercellular communication (GJIC) belongs among critical epigenetic events of tumor promotion. We determined the relative potencies of a series of environmentally relevant PCB congeners to inhibit GJIC in vitro in a rat liver epithelial cell line with pluripotent oval cell characteristics. The nonplanar PCBs were potent inhibitors of GJIC, whereas the coplanar PCBs did not inhibit GJIC. We then compared the effects of the coplanar PCB 126 (3,3',4,4',5-pentachlorobiphenyl) and the noncoplanar PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl) with effects of two model GJIC inhibitors, a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF). In contrast to TPA or EGF, PCB 153 elicited a long-term downregulation of GJIC (up to 48 h). Using Western blot analysis with phospho-specific antibodies, it was found that PCB 153, and not PCB 126, activated mitogen-activated protein kinases ERK1/2; however in contrast to TPA and EGF, this activation was observed at the time points subsequent to GJIC inhibition. Moreover, blocking of ERK1/2 activation did not prevent the GJIC inhibition induced by PCB 153. Therefore, additional intracellular signaling pathways potentially involved in the downregulation of GJIC by PCBs were screened by using specific chemical probes inhibiting serine/threonine kinases, tyrosine kinases, and phospholipases. The inhibition of diacylglycerol lipase partially blocked and the selective inhibition of Src kinases and phosphatidylcholine-specific phospholipase C (PC-PLC) completely blocked the inhibitory effects of the noncoplanar PCB on GJIC, indicating that PC-PLC or sphingomyelinase and Src might be upstream regulators of noncoplanar PCB-induced inhibition of GJIC.     

Apoptosis-mediated neurotoxic potential of a planar (PCB 77) and a nonplanar (PCB 153) polychlorinated biphenyl congeners in neuronal cell cultures

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic depending on the chemical structure of the congeners. This study investigated the neurotoxic potential of 3,3',4,4'-tetrachlorobiphenyl (TCB) (PCB 77, a non-ortho-substituted planar congener) and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) (PCB 153, a di-ortho-substituted nonplanar congener) by assessing cell viability and apoptotic cell death in neuronal cell cultures. We have combined morphological and biochemical techniques to establish the relevance of apoptosis in neuronal cell death induced by the two selected PCB congeners. Treatment with both planar and nonplanar congeners caused the loss of cell viability and accelerated apoptosis in a time- and concentration-dependent manner. However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (PCB 77) than for the di-ortho-substituted nonplanar congener (PCB 153). This was correlated with the loss of cell viability since the planar congener was more cytotoxic. Based on our findings, the apoptosis induced by PCBs involves the increase of caspase-3 activity in neuronal cell cultures. It is reasonable to assume that PCB-induced apoptosis may be linked to the neurotoxic effect of these toxicants and that different molecular mechanisms may operate in the induction of apoptosis depending on the planarity or nonplanarity of the PCB congeners.     

Aryl hydrocarbon receptor-activating polychlorinated biphenyls and their hydroxylated metabolites induce cell proliferation in contact-inhibited rat liver epithelial cells.

Polychlorinated biphenyls (PCBs) exhibit tumor-promoting effects in experimental animals. We investigated effects of six model PCB congeners and hydroxylated PCB metabolites on proliferation of contact-inhibited rat liver epithelial WB-F344 cells. The 'dioxin-like' PCB congeners, PCB 126, PCB 105, and 4'-OH-PCB 79, a metabolite of the planar PCB 77 congener, induced cell proliferation in a concentration-dependent manner. In contrast, the 'non-dioxin-like' compounds that are not aryl hydrocarbon receptor (AhR) agonists, PCB 47, PCB 153, and 4-OH-PCB 187, an abundant noncoplanar PCB metabolite, had no effect on cell proliferation at concentrations up to 10 muM. The concentrations of dioxin-like PCBs leading to cell proliferation corresponded with the levels inducing the expression of cytochrome P450 1A1 mRNA, suggesting that the release from contact inhibition was associated with AhR activation. The effects of PCB 126 and PCB 153 on expression of proteins controlling G0/G1-S-phase transition and S-phase progression were compared. Only PCB 126 was found to upregulate cyclin A and D2 protein levels, and to increase both total cyclin-dependent kinase 2 (cdk2) and cyclin A/cdk2 complex activities. Despite the observed upregulation of cyclin D2, no increase in cdk4 activity was observed. The expression of cdk inhibitor p27Kip1 was not affected by either PCB 126 or PCB 153. These results suggest that dioxin-like PCBs can induce cell proliferation of contact-inhibited rat liver epithelial cells by increasing cyclin A protein levels, a process that then leads to upregulation of cyclin A/cdk2 activity and initiation of DNA replication. This mechanism could be involved in tumor-promoting effects of dioxin-like PCBs.     

Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153

Exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4′,5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2′,3,4,4′,5′ pentachlorobiphenyl: PCB118), non-coplanar (2,2′,4,4′,5,5′-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter timepoints. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly different from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.     

Effects of structurally different noncoplanar and coplanar PCBs on HELF cell proliferation,cell cycle,and potential molecular mechanisms

Polychlorinated biphenyls (PCBs) are a group of chemicals that persist in the environment, indoors, and humans. Lung exposure to airborne and food contaminants, such as PCBs, may cause possible lung disorders, such as cancer. In the present study, we investigated the effects of structurally different lower chlorinated (≤4Cl), noncoplanar PCB40, and coplanar PCB77 on human lung fibroblast cell line (HELF) cell proliferation, cell cycle progression, and possible molecular mechanisms. Noncoplanar PCB40 and coplanar PCB77 exhibited concentration‐ and time‐dependent biphasic dose–response effects on HELF cell proliferation. Noncoplanar PCB40 and coplanar PCB77 induced 23 and 45% cytotoxicity at higher concentrations than the control. The flow cytometry analysis showed that exposure to PCB40 caused a significant increase in time spent in the G1 phase but decreased length of the S phase in a concentration‐ and time‐dependent manner, whereas PCB77 exposure decreased time spent in the G1 and S phases but increased time spent in the G2 phase. Western blot analysis indicated that PCB77 increased the expression of cyclin E, CDK2, p21, and caspase‐9, while PCB40 decreased the expression of these proteins (except CDK2 and p21). An increase in CDK expression after exposure to PCB77 suggests that it may cause carcinogenic effects on HELF cells at higher doses. Our results also demonstrate that the different cytotoxic effects induced by coplanar and nonplanar PCBs were correlated with their structural characteristics; the coplanar congener was more cytotoxic than the nonplanar congener. The study elaborates threshold levels for these chemicals and suggests that the cytotoxicity mechanisms by which PCB congeners act on HELF cells depend on their planarity and chemical structures. Furthermore, the study will be important for developing antidotes to the adverse effects and risk assessment practices for PCBs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1183–1190, 2017.     

Effect of PCB 126 and PCB 153 on incidence of apoptosis in cultured theca and granulosa cells collected from small,medium and large preovulatory follicles

The aim of the presented study was to evaluate the effects of PCB 126 and PCB 153 on granulosa and theca cell apoptosis. Granulosa and theca cells were collected from small, medium, and large preovulatory porcine follicles and cultured as monolayers. Cells were initially cultured for 24 h to allow attachment to the plates. Media were changed and 100 pg/ml PCB 126 or 100 ng/ml PCB 153 were added. After 48 h, granulosa and theca cells were fixed for assessment of the number of apoptotic cells utilizing a Hoechst staining technique or frozen for measurement of caspase-3 activity. Media were collected for testosterone concentration analysis from theca cell cultures or estradiol from granulosa cell cultures. Neither PCB 153 nor PCB 126 had an effect on testosterone secretion by theca cells collected from small and medium size follicles, while both PCBs decreased testosterone secretion by large follicles. The decrease in testosterone secretion by large follicles under the influence of both PCBs was paralleled by a suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. Neither of the PCBs had an effect on estradiol secretion by granulosa cells collected from small and medium size follicles, while both PCBs increased estradiol in granulosa cells collected from large follicles. PCB-associated increased estradiol secretion by granulosa cells collected from large follicles was accompanied by suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. In conclusion, we have presented evidence that in preovulatory follicles PCBs inhibit both theca and granulosa cells apoptosis. Therefore, an exposure to PCBs may cause alterations in the pattern of terminal differentiation of follicles and attenuate spontaneous elimination of atretic follicles.     

Responsiveness of hepatic and cerebral cytochrome P450 in rat offspring prenatally and lactationally exposed to a reconstituted PCB mixture

Perinatal polychlorinated biphenyl (PCB) exposures still remain a serious health concern because offspring receive PCB burden from mother during vulnerable processes of development. Since cytochrome P450 (CYP) represents a toxicological endpoint, in the present study, representing an extended investigation of a previous multitasked one, we explored the long‐term responsiveness of CYP1A and CYP2B isoforms by Western blot analysis in liver and whole brain of lactating (PN12), weaning (PN21), and adult offspring (PN60) rats prenatally and lactationally exposed to a reconstituted PCB mixture (RM) of noncoplanar PCB138, 153, 180, and coplanar PCB126 congeners. We chose highly chlorinated PCBs instead of lower chlorinated one, because their recalcitrance to biotransformation makes easy their accumulation/persistence in tissues and breast milk. Dioxin‐like congener PCB126 binding aryl hydrocarbon receptor (AHR) is responsible of many toxic effects. Pregnant Sprague–Dawley dams with high affinity AHR received subcutaneous injection of RM (10 mg/kg body weight) daily during gestation (days 15–19) and twice a week during breast‐feeding. The results evidenced a transfer of PCBs to neonates through milk and a significant responsiveness of hepatic CYP in both mothers and offspring. In liver of exposed progeny, CYP isoforms exhibited a significant increment at PN12 (70% over control) and at PN21 (270% over control). Contrary to dams, in adult PCB offspring CYP levels showed a decline up to values similar to those of control. This transient developmental responsiveness of CYP isoforms in offspring liver reflects roughly the time course of hepatic PCB levels previously reported. Even if congeners were detected in brain, we failed in evidencing a responsiveness of CYP isoforms probably because of region‐specific CYP expression in this organ. In conclusion, induction of offspring hepatic CYP is index of liver PCB burden, and despite the insensitivity of whole brain CYP we cannot exclude brain vulnerability toward PCB. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 856–866, 2014.     

Ortho-substituted PCBs kill cells by altering membrane structure.

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.     

Role of N-methyl-D-aspartate receptors in polychlorinated biphenyl mediated neurotoxicity

Polychlorinated biphenyls (PCBs) are widespread persistent environmental pollutants. Chronic human and animal exposure to PCBs results in various harmful effects including neurotoxicity. This study investigates the effects of the PCB mixture Aroclor 1254 (A1254) and two PCB congeners (coplanar, non-ortho PCB 126, and non coplanar PCB 99) on the expression of N-methyl-D-aspartate receptors (NMDARs) and the subsequent toxic effects using a human SHS5-SY neuroblastoma cell line. NMDAR was measured using a radiolabeled phencyclidine receptor ligand [³H]-MK801, apoptosis was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and cell death using lactate dehydrogenase (LDH) release. After treatment, a positive dose–response relationship of increasing NMDARS, increasing caspase-3 activity and cell death was observed in all PCB compounds. The non-coplanar PCB compounds were found to be significantly more toxic than the coplanar congener and the PCB mixture A1254. PCB-mediated cell death was attenuated with 10 μM NMDAR antagonists: 1-amino-3,5-dimethyladamantane hydrochloride (memantine) and (+)-5-methyl-10,11-dihydro-5H-debenzocyclhepten-5,10-imine maleate ((+)-MK-801), thus demonstrating the importance of NMDAR in PCB neurotoxicity. Intracellular calcium [Ca²⁺]_i chelator BAPTA-AM (1 μM) partially attenuated the neurotoxic effect of the PCBs suggesting a role of calcium homeostasis disruption in the neurotoxicity of PCBs. These results suggest that the neurotoxicity of PCBs can be mediated through activation of NMDARs.     

Effects of PCB 52 and PCB 77 on cell viability, [Ca(2+)](i) levels and membrane fluidity in mouse thymocytes

Exposure to polychlorinated biphenyls (PCBs) is known to suppress immune system function and this action is usually ascribed to dioxin-like PCBs that act via the Ah receptor. We have studied the effects of one ortho-substituted, non-dioxin-like PCB (PCB 52) and one coplanar, dioxin-like congener (PCB 77) on properties of thymocytes acutely isolated from mice. Viability of thymocytes was dose- and time-dependently reduced by PCB 52 with a threshold concentration of about 1 microM, while there was no effect of PCB 77 on viability at concentrations less than 10 microM. Cell death was detectible within 5 min of exposure. Both congeners caused a dose-dependent increase in [Ca(2+)](i), but the threshold concentration was 1 microM for PCB 52 and 5 microM for PCB 77. However, the cell death was not due to the elevation of [Ca(2+)](i), since it was not reduced by incubation in Ca-free Tyrode's Solution. PCB 52, but not PCB 77, caused an increase in membrane fluidity at a concentration of 5 microM. These observations are consistent with previous results that suggest that ortho-substituted PCB congeners dissolve in cell membrane and cause greater disruption of function than do dioxin-like PCB congeners.     

Exposure to the Three Structurally Different PCB Congeners (PCB 118, 153, and 126) Results in Decreased Protein Expression and Altered Steroidogenesis in the Human Adrenocortical Carcinoma Cell Line H295R

Polychlorinated biphenyls (PCB), synthetic, persistent organic pollutants (POP), are detected ubiquitously, in water, soil, air, and sediments, as well as in animals and humans. PCB are associated with range of adverse health effects, such as interference with the immune system and nervous system, reproductive abnormalities, fetotoxicity, carcinogenicity, and endocrine disruption. Our objective was to determine the effects of three structurally different PCB congeners, PCB118, PCB 126, and PCB 153, each at two concentrations, on the steroidogenic capacity and proteome of human adrenocortical carcinoma cell line cultures (H295R) . After 48 h of exposure, cell viability was monitored and estradiol, testosterone, cortisol and progesterone secretion measured to quantify steroidogenic capacity of the cells. Two-dimensional (2D) gel-based proteomics was used to screen for proteome alterations in H295R cells in response to the PCB. Exposure to PCB 118 increased estradiol and cortisol secretion, while exposure to PCB 153 elevated estradiol secretion. PCB 126 was the most potent congener, increasing estradiol, cortisol, and progesterone secretion in exposed H295R cells. Seventy-three of the 711 spots analyzed showed a significant difference in normalized spot volumes between controls (vehicle only) and at least one exposure group. Fourteen of these protein spots were identified by liquid chromatography with mass spectroscopy (LC-MS/MS). Exposure to three PCB congeners with different chemical structure perturbed steroidogenesis and protein expression in the H295R in vitro model. This study represents an initial analysis of the effects on proteins and hormones in the H295R cell model, and additional studies are required in order to obtain a more complete understanding of the pathways disturbed by PCB congeners in H295R cells. Overall, alterations in protein regulation and steroid hormone synthesis suggest that exposure to PCB disturbs several cellular processes, including protein synthesis, stress response, and apoptosis.     

In vivo and in vitro effects of PCB126 and PCB153 on rat testicular androgenesis

In this study we compared the effects of PCB126 and PCB153 on adult rat testicular androgenesis and the status of antioxidant enzymes in the interstitial cell compartment 96h after local intratesticular application. Obtained results indicated PCB126-induced inhibition of conversion of progesterone (P) and Δ(4)-androstenedione (A(4)) to testosterone (T), and stimulation of conversion of P to T induced by PCB153, while combined application had no effect. Activities of antioxidant enzymes were unchanged, except of decreased activity of SOD in PCB126-treated group. In parallel experiments, adult purified Leydig cells challenged with PCB congeners were incubated for 2h in the presence of corresponding steroid substrates. Results demonstrated that in the presence of subsaturating substrate concentrations PCB126 induced inhibition of conversion of P and A(4) to T at nM to μM doses, while PCB153 caused stimulation at nM concentrations. Further studies should indicate possible mechanism(s) of modulation of androgenesis by tested PCB congeners.     

Different effects of PCB101, PCB118, PCB138 and PCB153 alone or mixed in MCF-7 breast cancer cells

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that can be a potential health hazard. In the present study we analyzed the potential estrogenic effect in MCF-7 cells of four biologically relevant PCB congeners, alone or in mixtures, present in dairy products, vegetable oil and fish: PCB101, PCB118, PCB138 and PCB153. The mixture of four PCB was tested at seven different concentrations. We investigated the ability of these PCBs, alone or mixed, to induce cell proliferation, and the level of estrogen-regulated protein pS2, in human MCF-7 breast cancer cells. PCB153 (35 microM) stimulated cell proliferation from 48 h up to day 6, PCB118 (40 microM) only at 48 h, but PCB101 (45 microM) and PCB138 (15 microM) applied to the cells for 6 days had no effect. In contrast, the various concentrations of mixtures significantly reduced cell proliferation at different times. No change in pS2 levels was seen after treatment with the PCBs alone or mixed. In exploring the mechanism of these events, we found that PCB153 induced mitogen-activated protein kinase (MAPK) ERK1/2 at 4, 8 and 12 h, while the antiproliferative effect seemed to be related to an apoptotic action beginning at 12 h and ending at 48 h. These findings indicate that these PCBs alone or mixed have no estrogenic effect in MCF-7 cells, although PCB153 induce an ERK1/2-mediated mitogenic effect. On the contrary the mixture of PCBs induces an antiproliferative effect, ascribable to an apoptotic action.     

Coplanar and non-coplanar congener-specificity of PCB bioaccumulation and immunotoxicity in sea stars

The sea star Asterias rubens (L.), a representative species of the North Sea benthic environment, was exposed to a mixture of 10 selected PCB congeners (3 coplanar or c-PCBs, and 7 non-coplanar) via experimentally contaminated sediments. Both the degree of bioaccumulation and subsequent immunotoxic effects of these PCBs were determined. A strong congener-specificity for both bioaccumulation and immunotoxicity was found as well as a probable induction of a congener-specific detoxification mechanism resulting in the dramatic decrease in body levels of the three coplanar congeners tested (PCBs 77, 126 and 169). Moreover, a correlation was found between the bioaccumulation of c-PCBs and their immunotoxic effects. These findings suggest that coplanar congeners should be included in the list of congeners recommended to be analyzed for biological impact-oriented marine monitoring programmes.     

PCB 9 exposure induces endothelial cell death while increasing intracellular calcium and ROS levels

Cultured porcine endothelial cells were used to determine the effects of several congeners of polychlorinated biphenyls (PCBs) on cell viability and changes induced by these congeners on levels of intracellular calcium and reactive oxygen species (ROS). Cultured endothelial cells were prepared as a single suspension and run on flow cytometry. Cell viability, intracellular calcium, and ROS concentrations were simultaneously determined by using propidium iodide, Fluo-3, and dihydrorodamine, respectively. The congeners tested included two coplanar tetrachlorinated congeners that have no ortho chlorines, PCBs 77 and 80, a tri-chloro congener which does not have any ortho chlorine, PCB 39, a di-ortho, tetrachlorinated congener, PCB 52, and a mono-ortho congener, PCB 9. PCB 9 was cytotoxic at 5 μM within 5 min of exposure, and the toxicity increased with time and concentration. None of the other congeners showed consistent cytotoxicity. The cytotoxicity was roughly correlated with elevations in cellular ROS levels, but not with changes in intracellular calcium. To the best of our knowledge, toxicity of lower chlorinated, more volatile congeners such as PCB 9 has not been previously studied. These observations may be taken to explain the elevated risk of cardiovascular disease previously reported among residents living near to hazardous waste sites containing PCBs.     

Polychlorinated biphenyl-induced apoptosis of murine spleen cells is aryl hydrocarbon receptor independent but caspases dependent

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants and many of their toxic effects, including their immunotoxicities, are mediated by the activation of aryl hydrocarbon receptor (AhR). We previously reported that Aroclor 1254, one of the most widely used PCB mixtures, increased DNA fragmentation in mouse spleen cells, suggesting that apoptosis was correlated with the immunotoxicity of PCB (Yoo et al., Toxicol. Lett. 91, 83-89, 1997). In the present study we investigated the mechanism by which PCB induces apoptosis and the involvement of AhR in the PCB-mediated apoptosis of mouse spleen cells. Aroclor 1254 induced DNA fragmentation without AhR activation, and the apoptosis was unaffected by alpha-naphtoflavone, a well-known antagonist of AhR. Moreover, the PCB congeners (PCB 47, 52, 128, and 153), which have little affinity for AhR, induced DNA fragmentation, whereas congeners (PCB 77, 126, and 169) that have high affinity for AhR did not induce fragmentation. The di-ortho form of PCB (PCB 153) and Aroclor 1254 induced DNA fragmentation in the spleen cells of both AhR knockout mice and Ah low-response mice, whereas the non-ortho form of PCB (PCB 126) did not induce DNA fragmentation. In the light of these findings, it is evident that AhR is not involved in PCB-mediated apoptosis. PCB 153 significantly increased caspase-3 activity in both spleen cells and human leukemia cells, and z-VAD-fmk, a general inhibitor of caspases, prevented PCB-induced DNA fragmentation. Based on our findings, the most likely mechanism that can account for this biological effect involves the induction of caspase-dependent apoptotic cell death.