Immunosuppressive Activity of Polychiorinated Biphenyl Mixtures and Congeners: Nonadditive (Antagonistic) Interactions

The dose-response inhibition of the splenic plaque-forming cell(PFC) response and serum IgM units to the antigen, trinitrophenyl-lipopolysaccharide,was determined for several polychiorinated biphenyl (PCB) mixturesand congeners in female B3C3F1 mice. The ED50 values for Aroclor1260-, 1254-, 1248-, and 1242-induced immunotoxicity variedby less than twofold from 355 to 699 mg/kg. The range of ED50values for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl, 3,3' ,4,4',5-pentaCB, 3,3',4,4',5,5'-hexaCB,2,3,3',4,4'-pentaCB, 2,3',4,4',5-pentaCB, 2,3,3',4,4',5-hexaCB,2,3,3',4,4',5,5'-heptaCB, 2,2',3,3',4,4',5-heptaCB, and 2,2',3,4,4',5,5'-heptaCBwere 4.6 to 4.9, 134 to 245, 4.7 to 7.0, 6.9 to 11.1, 88,000to 121,000, 122,000 to 132,000, 99,000 to 157,000, 89,000 to129,000, 117,000 to 240,000, and 132,000 to 238,000 µg/kg,respectively. The immunotoxicity-derived toxic equivalency factors(TEFs) for these congeners could be calculated from the ED50(TCDD)/ED50 (congener) ratios and the TEF values were withinthe range of those previously determined for other aryl hydrocarbonreceptor-mediated responses. Based on the known concentrationsof these congeners in the PCB mixtures, TCDD or toxic equivalents(TEQs) in the mixture were calculated [i.e., TEQ = (PCB_congenerx TEF)] using the immunotoxicity-derived TEFs (plaque-formingcells/10⁶ viable cells). TEQ values for Aroclors 1260, 1254,1248, and 1242 were 16.0, 54.4, 260.4, and 197 ppm, respectively.Based on the ED50 value for the immunosuppressive activity ofTCDD (4.8 µg/kg), the calculated ED50 values for immunesuppression by Arodors 1260, 1254, 1248, and 1242 were 300,88, 18, and 24 mg/kg, respectively. The ED50 (observed)/ED50(calculated) ratios were 1.2, 5.9, 21, and 22.0 for Aroclors1260, 1254, 1248 and 1242, respectively. Thus, for Aroclors1254, 1248, and 1242, the high ED50 (observed)/ED50 (calculated)ratios (i.e., 5.9 to 22.0) indicate that the TEF approach overestimatesthe toxicity of these mixtures due to non-additive (antagonistic)interactions of the PCBs. In contrast, the TEF approach wasuseful in determining the immunotoxicity of the Aroclor 1260mixture.     

Differential in vitro neurotoxicity of the flame retardant PBDE-99 and of the PCB Aroclor 1254 in human astrocytoma cells

Polybrominated diphenyl ethers (PBDEs) are an important class of flame retardants. Because of their presence in maternal milk and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible developmental neurotoxicity. Aim of the present study was to investigate the in vitro effects of PBDE-99 (2,2', 4,4', 5-pentabromodiphenyl ether) on astroglial cells (human 132-1N1 astrocytoma cells) and comparing it with those of the PCB mixture Aroclor 1254. Both PBDE-99 and Aroclor 1254 caused a concentration-dependent inhibition of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction, however, only the latter increased lactate dehydrogenase (LDH) release or cell death, assessed by the trypan blue assay. PBDE-99 caused translocation of the three protein kinase C (PKC) isozymes (alpha, epsilon, zeta) present in 132-1N1 astrocytoma cells, while Aroclor 1254 affected only PKCalpha and epsilon translocation. However, pre-incubation with the PKC inhibitor GF109203X or PKC down-regulation by the phorbol ester PMA, had minimal or no effect on PBDE-99 or Aroclor 1254-induced cytotoxicity. Similarly, the calcium chelator BAPTA-AM, the tyrosine kinase inhibitor genistein, and the MEK (mitogen activated protein kinase kinase) inhibitor PD98059 had no effect on PBDE-99 and Aroclor 1254 cytoxicity. On the other hand, the phosphatidylinositol 3 kinase (PI-3K) inhibitor LY290042 enhanced PBDE-99 toxicity, but did not affect Aroclor 1254. Because of the involvement of PI-3K in apoptotic cell death, the ability of PBDE-99 and Aroclor 1254 to induce apoptosis in astrocytoma cells was investigated. PBDE-99, but not Aroclor 1254, caused apoptotic cell death in astrocytoma cells, assessed by the TUNEL method and by Hoechst 33258 staining, via a p53 dependent mechanism. These results suggest that PBDE-99 and Aroclor 1254 exert differential cytotoxic effects on human astroglial cells.     

Heme-oxygenase-1 promotes polychlorinated biphenyl mixture aroclor 1254-induced oxidative stress and dopaminergic cell injury.

Dopaminergic (DAergic) systems have been identified as putative targets for polycholorinated biphenyl (PCB) actions. However, the precise mechanisms leading to neurotoxicity are unresolved. Reactive oxygen species (ROS) were recently shown to mediate injury in DAergic MN9D cells following exposure to Aroclor 1254 (A1254), a commercial PCB mixture. The oxidative stress response in DAergic cells included a persistent expression of heme oxygenase-1 (HO-1). This study tested the hypothesis that a sustained PCB-induced HO-1 response leads to abnormally high Fe levels, which generates ROS production and mediates death in the MN9D DAergic cell model. Accordingly, results indicated that A1254 augmented intracellular Fe levels in MN9D cells after 24 h. Fe chelation by desferoxamine or pharmacologic inhibition of HO activity with tin-protoporphyrin reduced Fe accumulation, ROS production, and cytotoxicity following A1254 exposure. HO-1 over-expression predisposed MN9D DAergic cells to enhanced ROS production and cell death in response to PCBs. Conversely, antisense inhibition of HO-1 expression prevented PCB-induced ROS production and cell death. These observations suggest that enhanced HO-1 catalytic activity and subsequent liberation of Fe participate in neurotoxic DAergic cell injury caused by A1254 exposure in vitro.     

The effect of polychlorinated biphenyls on the high affinity uptake of the neurotransmitters, dopamine, serotonin, glutamate and GABA, into rat brain synaptosomes

Studies have shown that polychlorinated biphenyls (PCB) may affect cognitive functions both in human and also in experimental animals. We have investigated whether this effect could be caused by an inhibition of the uptake of selected neurotransmitters into rat brain synaptosomes. Ortho-chlorinated biphenyls were found to inhibit transmitter transport into synaptosomes from rat brain. In contrast, several nonortho-chlorinated biphenyls did not inhibit uptake. The uptake of dopamine, glutamate, GABA and serotonin was inhibited by the PCB mixtures, Aroclor 1242 and 1254. Under identical condition, the uptake of dopamine was inhibited more efficient than that of glutamate. The inhibition of neurotransmitter uptake was found to be dependent on the chlorination patterns of the PCB congeners, (i) ortho-chlorinated PCBs with four to five chlorine substituents (with the exception of 2,2',6,6'-TeCB) were the most effective inhibitors; (ii) hexa- or heptachlorinated PCBs were poor inhibitors or partial inhibitors (e.g. 2,2',4,4',5,5'-HCB) of glutamate and GABA uptake. Kinetic studies indicated that Aroclor 1242 inhibited dopamine uptake mainly competitively. The uptake of glutamate and GABA was inhibited in either a mixed competitive or in a non-competitive way, respectively. The neurotoxic consequences of the effect of different PCBs on neurotransmitter uptake on the uptake into synaptosomes are discussed.     

Enhanced polychlorinated biphenyl lesions in Moloney leukemia virus-infected mice.

The hepatic toxicity produced by polychlorinated biphenyls (PCB) was enhanced in mice that were inoculated with an oncogenic virus, Moloney leukemia virus (MLV). Whenever there was neoplastic involvement of the spleen by MLV, the hepatic lesions produced by PCB were more pronounced than in those of non-MLV inoculated mice. Mice were exposed to PCB Aroclors, 1254, 1242, and 1221 for six months. Aroclors 1254 and 1242 were hepatotoxic with Aroclor 1254 causing death. Aroclor 1221 did not affect the mice. Liver weights in mice that were fed PCBs for six months and then maintained on a PCB-free diet for an additional three months were comparable with those of non-PCB exposed mice. These results suggest that the PCB-produced hepatic lesions (noncirrhotic) regenerate after removal of PCB from the diet. Polychlorinated biphenyls did not affect (promote or induce) the oncogenesis of MLV in this study.     

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.     

Dose-response immunotoxicities of commercial polychlorinated biphenyls (PCBs) and their interaction with 2,3,7,8-tetrachlorodibenzo-p-dioxin

The relative potencies of the commercial polychlorinated biphenyl (PCB) mixtures Aroclors 1260, 1254, 1248, 1242, 1016 and 1232 to inhibit the murine splenic plaque-forming cell response to sheep red blood cells was determined by dose-response treatment of C57BL/6 mice followed by logit plot analysis of the data. The ED50 values obtained for Aroclors 1260, 1254, 1248, 1242, 1016 and 1232 were 104, 118, 190, 391, 408 and 464 mg/kg or 0.28, 0.35, 0.66, 1.5, 1.5 and 2.0 mmol/kg, respectively. It was apparent that the higher chlorinated PCB preparations (Aroclors 1260, 1254 and 1248) were more potent than the lower chlorinated preparations (Aroclors 1242, 1016 and 1232). Previous studies have shown that the interaction of a subeffective dose of Aroclor 1254 (25 mg/kg) with an immunotoxic dose (3.7 nmol/kg) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in significant antagonism of the toxicity of the latter compound. Co-treatment of mice with a 25 mg/kg dose of all the commercial Aroclors and with a 50 mg/kg dose of a reconstituted PCB mixture (resembling a PCB extract from human milk) with TCDD (3.7 nmol/kg) showed that, with the exception of Aroclor 1232, all of the commercial PCBs and the reconstituted PCB mixture significantly antagonized the TCDD-mediated inhibition of the splenic plaque-forming cell response in C57BL/6 mice. The identities of the PCB congeners responsible for this antagonism and the mechanism of this process are unknown and are currently being investigated.     

In vitro cytotoxicity of polychlorinated biphenyls (aroclors 1016, 1242, 1254 and 1260) and their effect on phospholipid and neutral lipid composition of Chinese hamster ovary (CHO-K1) cells

Cytotoxicity of 4 Aroclors (1016, 1242, 1254 and 1260) was compared in Chinese hamster ovary (CHO-K1) cells in Ham's F-12 medium. When parameters of toxicity were cell numbers or tissue protein, 50% lethality occurred at Aroclor concentrations between 30 and 45 ppm. An in vitro clonal assay with CHO-K1 cells was a sensitive indicator of cytotoxicity of the polychlorinated biphenyls (PCBs). From EC₅₀ values (concentration that allowed 50% survival of formed colonies), cytotoxicity was lower with Aroclor 1016 (32 ppm) and higher with Aroclors 1254 (27 ppm) and 1260 (28 ppm). In cells exposed 24 h to a marginally cytotoxic dose (20 ppm) of each Aroclor, phospholipid (PL) thin-layer chromatography (TLC) showed an increase in phosphatidylcholine (PC) and a decrease in phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG). Neutral lipid (NL) TLC of cells given Aroclors 1242, 1254 or 1260 showed a 3–4-fold increase in triglyceride (TG) and a similar reduction in cholesteryl esters (CE); in contrast to Aroclor 1016 which produced no change in TG and a smaller (2-fold) reduction in CE. Cholesterol and free fatty acid fractions were unaffected by any of the Aroclors. The TG:PL ratio remained unchanged in cells given Aroclor 1016, but increased 3–4-fold with Aroclors 1242, 1254, or 1260. Compared to total values in the untreated controls, CHO-K1 cells contained less neutral lipid and more phospholipid only with Aroclor 1016.These results support the concept that differences in the behavior of Aroclor 1016 are related to its PCB composition. Changes in membrane PL and NL components, observed at marginally cytotoxic levels of each Aroclor, provided further evidence that the PCBs may affect membrane integrity and associated metabolic functions.     

Differential effects of PCBs on the induction of apoptosis machinery and PKCalpha translocation in rat renal tubular cell cultures

We have demonstrated previously [Pérez-Reyes, P.L., Sánchez-Alonso, J.A., López-Aparicio, P., Recio, M.N., Pérez-Albarsanz, M.A., 2001. Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures. Biosci. Rep. 6, 765-778] that the polychlorinated biphenyls (PCBs) cause loss of cell viability and accelerate apoptosis in cell kidney cultures. Further investigations are necessary to elucidate the mechanism of apoptosis induction. In this way, we have analyzed in the present work the effects of PCBs on protein kinase C (PKC, a protein family intimately involved in the regulation of cell survival) and the expression of two proapoptotic (caspase-3 and Bax) and one antiapoptotic (Bcl-2) proteins. Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, a di-ortho-substituted nonplanar congener) and PCB 77 (3,3',4,4'-tetrachlorobiphenyl, a non-ortho-substituted planar congener), significantly increased PKCalpha activity compared to control cells in the cytosolic and particulate cell fractions, and increased the PKCalpha protein content in the particulate fraction. The nonplanar PCB 153 showed stronger effects than the coplanar congener PCB 77. In addition, Aroclor 1248 decreased both, procaspase-3 levels and the Bcl-2/Bax protein ratio. These findings indicate that PCBs, particularly nonplanar congeners, can induce apoptosis in primary renal tubular cells through the PKCalpha, caspase-3 and Bcl-2/Bax pathway.     

Ortho-substituted but not coplanar PCBs rapidly kill cerebellar granule cells.

Several PCB congeners were assessed for their cytotoxicity on cerebellar granule cells in an attempt to compare their structure-activity relationship as potential neurotoxicants and to assess the mechanisms associated with their toxicity. Flow cytometry was used to monitor the changes of a number of biochemical endpoints: membrane integrity, intracellular free calcium concentration ([Ca(2+)](i)), reactive oxygen species (ROS) production, mitochondrial membrane potential (Delta psi(m)), and cell size. The non-coplanar, ortho-substituted congeners, PCB 8 (2,4'-dichlorobiphenyl), PCB 28 (2,4,4'-trichlorobiphenyl), PCB 47 (2,4,2',4'-tetrachlorobiphenyl), and PCB 52 (2,5,2',5'-tetrachlorobiphenyl) (10 microM) killed neurons to different degrees within 30 min. Loss of viability was accompanied by increased [Ca(2+)](i) and decreased Delta psi(m). No significant changes of ROS level were observed during exposure. The coplanar congeners, PCB 77 (3,4,3',4'-tetrachlorobiphenyl), PCB 80 (3,5,3',5'-tetrachlorobiphenyl), and PCB 81 (3,4,5,4'-tetrachlorobiphenyl) (10 microM), had no effects on membrane integrity, [Ca(2+)](i) or Delta psi(m) in this time period of exposure. In Ca(2+)-free Tyrode's medium, there was no [Ca(2+)](i) increase after exposure to the ortho-substituted congeners, but also no reduction in loss of membrane integrity, suggesting Ca(2+) influx was not the cause of viability loss. The mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (1-2 microM), caused a large decrease of Delta psi(m), but only a slight loss of viability, which suggested that Delta psi(m) is not the primary cause of PCB 52-induced cell death. These studies show that ortho-substituted PCBs are toxic to cerebellar granule cells; however, their toxic action is not secondary to elevation of intracellular calcium, a change in mitochondrial membrane potential, or free radical generation.     

Effects of polychlorinated biphenyls on the neutrophil NADPH oxidase system

Polychlorinated biphenyls (PCBs) are reported to induce the formation of reactive oxygen species (ROS) in human neutrophil granulocytes through the activation of the NADPH oxidase. The purpose of the present study is to elucidate the cellular mechanisms responsible for the activation of the NADPH oxidase after exposure to PCB. We have previously shown that PCB activates human neutrophil granulocytes through a calcium dependent activation of phospholipase D and/or phospholipase C, followed by the activation of protein kinase C. In the present study, pharmacological characterization of Aroclor (A) 1242-induced respiratory burst in human neutrophils was conducted by the use of enzymatic inhibitors. Pre-incubation with U0126, SB203580, SP600125, cyclosporin A and FK506 attenuated the A 1242-induced respiratory burst, measured by DCF-fluorescence, and luminol-amplified chemiluminescence. Our results show that the Erk1/2 kinases and p38MAPK/JNK are involved in ROS formation in neutrophils exposed to A 1242.     

Kinetic parameters of L-[125I]triiodothyronine degradation in rats pretreated with polyhalogenated biphenyls

These studies were designed to elucidate the effects on thyroid function and thyroid-hormone activity of the long-term administration of low doses of four polyhalogenated aromatic compounds. The compounds studied were the polychlorinated biphenyls (PCBs) Aroclor 1254 and Aroclor 1242, and the polybrominated biphenyls (PBBs) hexabromobiphenyl and octabromobiphenyl. Groups of eight female Sprague-Dawley rats, specified pathogen free, were fed a diet containing 50 ppm of one of the polyhalogenated biphenyls or control diet for 7 months. Significant effects on serum triiodothyronine (T3) were observed in the group given Aroclor 1254. Analysis of kinetic data revealed a decrease in T3 degradation rate and an increase of biological half-life after long-term exposure to Aroclor 1254. The T3 distribution space was increased in both groups treated with PCB, suggesting that PCB intoxication may open additional fluid pools to T3, possibly because of cell damage. Analysis of values for the same parameters for rats treated with PBBs showed less marked effects. The results of this study indicate that PCBs exert a direct effect on the thyroid gland and that the rate of synthesis of T3 may be reduced, and suggest that the mechanism of thyroid-hormone synthesis may be impaired.     

Percutaneous absorption and skin decontamination of PCBs: in vitro studies with human skin and in vivo studies in the rhesus monkey

Knowledge of the entry of polychlorinated biphenyls through the skin into the body and subsequent disposition aids estimation of potential for human health hazard. [14C]Aroclor 1242 and [14C]Aroclor 1254 were separately administered intravenously and topically to rhesus monkeys. Following iv administration, 30-d excretion was 39.4 +/- 5.9% urine and 16.1 +/- 0.8% feces (total 55.5 +/- 5.1%) for Aroclor 1242, and 7.0 +/- 2.2% urine and 19.7 +/- 5.8% feces (total 26.7 +/- 7.5%) for Aroclor 1254. Mineral oil and trichlorobenzene are common PCB cosolvents in transformers. Skin absorption of Aroclor 1242 was 20.4 +/- 8.5% formulated in mineral oil and 18.0 +/- 3.8% in trichlorobenzene (p greater than .05). Absorption of Aroclor 1254 was 20.8 +/- 8.3% in mineral oil and 14.6 +/- 3.6% in trichlorobenzene (p greater than .05). PCBs are thus absorbed through skin, and excretion from the body is slow. Vehicle (trichlorobenzene or mineral oil) did not affect percutaneous absorption. In vitro skin absorption in human cadaver skin did not correlate with in vivo findings. This was due to lack of PCB partition from skin into the water receptor fluid, even with addition of 6% Oleth 20 (Volpo 20) solubilizer. Skin decontamination of PCBs showed soap and water to be as effective as or better than the solvent ethanol, mineral oil, and trichlorobenzene in removing PCBs from skin. There is a dynamic time lapse for PCBs between initial skin contact and skin absorption (irreversible removal). Thus initially most PCBs could be removed from skin, but this ability decreased with time to the point where at 24 h only about 25% of the initial PCB skin dose could be recovered with skin washing.     

Effects of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) on thyroid hormone and vitamin A levels in rats and mice

The ability of the commercial polybrominated diphenyl ether (PBDE) preparation Bromkal 70-5 DE to alter thyroid hormone and vitamin A levels as well as microsomal enzyme activities was compared with that of the commercial polychlorinated biphenyl (PCB) preparation Aroclor 1254 in orally exposed female rats (Sprague-Dawley) and mice (C57BL/6 N). Additional mice were exposed to the PBDE congener 2,2',4,4'-tetrabromodiphenyl ether (DE-47), or to the PCB congener 2,3,3',4,4'-pentachlorobiphenyl (CB-105). For 14 days the animals were given approximately isomolar daily oral doses of Aroclor 1254, CB-105 (both 10 mg/kg body weight), Bromkal 70-5 DE or DE-47 (both at 18 mg/kg body weight). In addition, further groups of rats and mice received a higher dose of Bromkal 70-5 DE, 36 mg/kg body weight. Bromkal 70-5 DE and DE-47 decreased plasma free and total thyroxine (T4) levels in both rats and mice, although with lower potency than that of Aroclor 1254 and CB-105. By contrast, thyroid-stimulating hormone (TSH) levels were not significantly changed in any of the groups. Reduction of hepatic vitamin A levels was seen in rats after Aroclor 1254 and Bromkal 70-5 DE exposure. A similar tendency was seen also in mice, but the effects were significant only for concentration data and not the total amount. Induction ofmicrosomal phase I enzymes, measured as ethoxy, methoxy and pentoxy resorufin O-dealkylase (EROD, MROD, PROD) activities, was greatest after exposure to Aroclor 1254/CB-105 but were also significant in the Bromkal 70-5 DE/DE-47-treated groups. However, induction of uridine diphosphoglucuronosyl transferase (UDPGT) was small and for most groups insignificant. In conclusion, the PBDE compounds studied, although having a lower potency than the PCB compounds, decreased thyroxine and vitamin A levels and induced microsomal enzyme activities. Rats were more sensitive to the observed effects than mice. Microsomal phase I activity might be related, directly or indirectly, to the T4 and vitamin A effects, whereas several factors (such as weak enzyme induction and lack of correlation with altered T4 and vitamin A levels) argue against any UDPGT-related effects.     

Comparative Carcinogenicity in Sprague-Dawley Rats of the Polychlorinated Biphenyl Mixtures Aroclors 1016, 1242, 1254, and 1260

A comprehensive chronic toxicity and carcinogenicity study wasconducted on a series of Aroclors (1016, 1242, 1254, and 1260).Each Aroclor was assessed at multiple dietary concentrations,ranging from 25 to 200 ppm, for 24 months in male and femaleSprague-Dawley rats. Liver toxicity was indicated by elevatedserum enzyme activity (AST, ALT, and GGT), elevated serum cholesterolconcentration, decreases in hematologic parameters (RBC, Hb,and Hct), hepatocellular hypertrophy, an increased incidenceof altered hepatocellular foci, and an increased incidence ofhepatocellular neoplasms (primarily adenomas). Liver toxicitywas distinctly more severe in females than in males. The incidenceof hepatocellular neoplasms was highly sex-dependent (females> males), differed between Aroclor mixtures and, for females,increased with dose and followed the general incidence patternof Aroclor 1254 > Aroclor 1260 Aroclor 1242 > Aroclor1016. A significant response (p < 0.05) in males was seenonly for the high dose of Aroclor 1260. A small increase inthe incidence of thyroid gland follicular cell adenomas wasnoted in males for Aroclors 1242, 1254, and 1260, with the incidencebeing uniform across dose groups and Aroclor mixtures. For females,increased survival relative to controls was observed for allAroclor treatment groups. A significantly decreased trend inthe incidence of mammary gland neoplasms compared to controlwas also noted for females receiving Aroclors 1242, 1254, and1260.     

Induction of cytochrome P450 activities by polychlorinated biphenyls in isolated mouse hepatocytes. Influence of Ah-phenotype and iron.

Exposure of cultured primary hepatocytes from Ah-responsive male C57BL/10ScSn mice to a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) at 0.1-20 micrograms/mL for up to 96 hr induced cytochrome P4501AI-mediated activity (ethoxyresorufin O-deethylase, EROD) up to 50-fold. In contrast, pentoxyresorufin O-dealkylase (PROD), which in some circumstances is a measure of phenobarbitone-induced cytochrome P450 isoenzymes, was induced only 5-fold. There were similar findings on EROD activities with the pure compounds 3,3',4,4',5,5'-hexachlorobiphenyl, 3,3',4,4',5,5'-hexabromobiphenyl and 3,3',4,4'-tetrachlorobiphenyl(TCB) and also beta-naphthoflavone but not with 2,2',4,4'-TCB or phenobarbitone. The higher concentrations of Aroclor 1254 were also associated with cytotoxicity as estimated by release of alanine aminotransferase (ALT) into the medium. Unlike in C57BL/10ScSn hepatocytes induction of EROD and cytotoxicity was minimal in hepatocytes from the Ah-non-responsive strain DBA/2. Although in vivo the hepatic toxicity and carcinogenicity of polyhalogenated aromatics are markedly potentiated by iron, no enhancement of the cytotoxicity of Aroclor 1254 towards C57BL/10ScSn hepatocytes by iron was observed in vitro. However, iron caused decreased EROD activities and possibly cytochrome P4501AI (as judged by Western blotting) as in vivo. Even in the presence of iron and the haem precursor 5-aminolaevulinic acid (5-ALA) there was no development of uroporphyria in this system although this occurs with Aroclor in vivo and is enhanced by iron. Accumulation of uroporphyrin did occur after extended culture of C57BL/10ScSn hepatocytes on matrigel for 8 days in the presence of 5-ALA and Aroclor 1254 but again no potentiation by iron was observed. Thus, although culture of Ah-responsive and -non-responsive hepatocytes mimics some aspects of the mechanisms of in vivo toxicity of PCBs, there is some unknown associated influence of iron metabolism which cannot, as yet, be produced in vitro but which is of importance in vivo.     

Elevation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) polychlorinated biphenyls. Structure-activity relationships

Administration of the commercial polychlorinated biphenyl (PCB) Aroclor 1254 to immature male Wistar rats resulted in increased levels (80-110%) of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) hepatic cytosolic receptor protein which remained elevated for 14 days. The effects of structure on the activity of individual PCB congeners to modulate hepatic cytosolic receptor levels were compared to the structure-activity relationships (SARs) which have been developed previously for PCBs as inducers of hepatic microsomal monooxygenases. 3,3',4,4'-Tetra- and 3,3',4,4',5-pentachlorobiphenyl induced the cytochrome P-448-dependent monooxygenase, ethoxyresorufin O-deethylase (EROD), and resembled 3-methylcholanthrene in their mode of monooxygenase enzyme induction. These congeners also bound to the receptor protein; however, neither compound increased hepatic cytosolic receptor protein levels. Several PCB congeners which exhibit low binding affinities for the cytosolic receptor protein resembled phenobarbitone (PB) in their mode of monooxygenase enzyme induction and, like PB, elevated cytosolic receptor protein levels. Nevertheless, a comparison of the time course of monooxygenase enzyme induction and receptor protein elevation by 2,2',4,4',5,5'-hexachlorobiphenyl and PB illustrated significant differences in their activities. PB-mediated elevation of receptor levels was maximized 24 hr after the last dose, and 48 hr later the receptor levels decreased to control values. In contrast, 5 days after administration of a single dose of 2,2',4,4',5,5'-hexachlorobiphenyl (300 mumoles/kg) the receptor levels were elevated significantly, and these increased levels (205-127% increases over control) persisted for 14 days. There was no correlation between increased levels of hepatic receptor protein and the induction of the cytochrome P-450-dependent monooxygenases, aldrin epoxidase or 4-dimethylaminoantipyrine N-demethylase. Two PCBs, 2,3,3',4,4',5- and 2,2',3,4,4',5-hexachlorobiphenyl, which resembled Aroclor 1254 in their mode of monooxygenase enzyme induction, also elevated hepatic receptor protein levels but were less active than the PB-type inducers. Thus, the SARs developed for PCBs which elevate cytosolic receptor levels demonstrate that the most active compounds exhibit the lowest affinity for the receptor protein and do not induce EROD. In contrast, the more toxic PCB congeners which are approximate isostereomers of 2,3,7,8-TCDD both induced EROD and bound with high affinity to the receptor protein but did not increase hepatic cytosolic receptor protein levels.     

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.     

Effects of PCB99 and PCB153 exposure on spermatogenesis in young adult C57BL6 mice

This study examined the effects of acute exposure to PCB99 (2,2',4,4',5-pentachlorobiphenyl), and PCB153 (2,2',4,4'5,5'-hexachlorobiphenyl), on spermatogenesis in 8-week-old C57BL6 mice. The mice were randomly allocated to PCB99 and PCB153 and a single dose of respectively 10 and 100 mg/kg was given by oral gavage. During the 6-week experiment, six mice per treatment group were sacrificed weekly, body weights were recorded and samples with respect to the male reproductive system were collected until further analysis. None of the treatments, showed changes in body weight or reproductive endpoints. Flow cytometric analysis revealed spermatogenesis to be unaffected. However, PCB99 and PCB153 showed a significant increase in Leydig cell apoptosis. The results from the present study indicate that the male reproductive system is relatively refractory to PCB99 and PCB153 at levels exceeding those of wildlife and humans, when exposed during adult life. However, the finding of apoptotic Leydig cells merits further investigation.     

Low-frequency hearing loss following perinatal exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in rats.

Previous research has demonstrated the sensitivity of the developing rat to the ototoxic effects of exposure to Aroclor 1254. In this study we assessed the effects of developmental exposure to an individual PCB congener (3,3',4,4',5-pentachlorobiphenyl; PCB 126) on auditory function. Nulliparous Long Evans rats received either 0, 0.25, or 1.0 microg/kg/day (5 days/week) for 35 days prior to breeding and throughout gestation and lactation. Auditory thresholds for 0.5-, 1-, 4-, 8-, 16-, 32-, and 40-kHz tones were assessed in offspring on postnatal days (PND) 76-90. Perinatal maternal PCB 126 exposure caused low-frequency hearing deficits. Elevated auditory thresholds occurred in the 1.0 microg/kg/day treated group for 0.5- and 1-kHz tones, whereas thresholds were not significantly affected at any higher frequencies. These results are important in that the data implicate, at least partially, the coplanar PCBs in the developmental ototoxicity induced by Aroclor 1254.