Inhibition of 3,3′,4,4′,5-Pentachlorobiphenyl-Induced Chicken Embryotoxicity by 2,2′,4,4′,5,5′-Hexachlorobiphenyl

3,3′,4,4′,5-Pentachlorobiphenyl (pentaCB) caused a dose-dependent induction of chicken embryolethality, malformations, edema, and liver lesions at doses ranging from 0.5 to 12.0 μg/kg. In contrast, no embryotoxicity was observed after treatment with 10, 25, or 50 mg/kg 2,2′,4,4′,5,5′-hexaCB. In eggs cotreated with 2.0 μg/kg, 3,3′,4,4′,5-pentaCB plus 10, 25, or 50 mg/kg 2,2′,4,4′,5,5′-hexaCB, there was significant protection from 3,3′,4,4′,5-pentaCB-induced embryo malformations, edema, and liver lesions, whereas no inhibition of embryolethality was observed. These results further extend the response-specific nonadditive interactions of binary mixtures of polychlorinated biphenyls (PCBs) and should be considered in the development of approaches for hazard assessment of PCB mixtures and related compounds.     

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.     

Low Inducibility of CYP1A Activity by Polychlorinated Biphenyls (PCBs) in Flounder (Platichthys flesus): Characterization of the Ah Receptor and the Role of CYP1A Inhibition

Several studies have reported a low inducibility of hepaticcytochrome P4501A (CYP1A) activity in European flounder (Platichthysflesus) following exposure to mixtures of polychlorinated biphenyls(PCBs). Here we report on mechanistic studies toward understandingthis low CYP1A inducibility of flounder, involving molecularcharacterization of the Ah receptor (AhR) pathway as well asinhibition of the CYP1A catalytic activity by PCB congeners.Hepatic cytosolk AhR levels in flounder were determined usinghydroxylapatite, protamine sulfate adsorption analysis, or velocitysedimentation on sucrose gradients. AhR levels in flounder ({smalltilde}2–7 fmol/mg protein) were much lower than observedgenerally in rodents ({small tilde}50–300 fmol/mg protein).Molecular characterization of the flounder AhR was providedby first-strand cDNA synthesis and amplification of flounderhepatic poly(A)⁺ RNA using RT-PCR. A 690-bp product was found,similar in size to a Fundulus AhR cDNA. The specificity of the690-bp band was established by Southern blotting and hybridizationwith a degenerate AhR oligonucleotide. The deduced amino acidsequence of the flounder AhR fragment was 59–60% identicalto mammalian AhR sequences. Although the AhR is present in floundercytosol, we were unable to demonstrate detectable amounts ofinducibk TCDD-AhR-DRE complex in gel-retardation assays. Highinduction levels of CYP1A protein and associated EROD activityhave been previously found in flounder following exposure to2,3,7,8-tetrachlorod-ibenzo-p-dioxin (TCDD). In contrast, theinduction of CYP1A catalytic activity by PCB mixtures remainsunexpectedly low. Therefore, we further characterized the inhibitorypotential of PCB congeners on CYP1A activity in flounder andcompared this with inhibitory effects of PCB congeners on ratCYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB,3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB,and the commercial PCB mixture Clophen A50 are potent competitiveinhibitors of hepatic microsomal CYP1A catalytic activity inflounder and rat The K_m for ethoxyresonifin (0.095 µM)in flounder is strikingly close to K_i's found for the testedPCBs. This emphasizes the possible involvement of PCB congenersin inhibition of EROD activity in PHAH exposed fish. Finally,our data indicate that flounder CYP1A is more efficient in metabolizingethoxyresonifin than that of rat CYP1A.     

Influence of Symmetrical Polychlorinated Biphenyl Isomers on Embryo and Fetal Development in Mice: II. Comparison of 4,4'-Dichlorobiphenyl, 3,3'4,4'-Tetrachlorobiphenyl, 3,3'5,5'-Tetrachlorobiphenyl, and 3,3'4,4'-Tetramethylbiphenyl

Outbred albino (CD-1) mice were given the following biphenylisomers by gavage in cottonseed oil on Days 6–15 of gestation:4,4'-dichlorobiphenyl (DCB) at 16, 32, and 64 mg/kg/day; 3,3',4,4'-tetrachlorobiphenyl(3,4-TCB) at 1, 2, 4, 8, 16, 32, and 64 mg/kg/day; 3,3',5,5'-tetrachlorobiphenyl(3,5-TCB) at 64 mg/ kg/day; and 3,3',4,4'-tetramethylbiphenyl(TMB) at 64 mg/kg/day. The mice were killed on Day 18 of gestation,necropsies were performed on the dams, and the fetuses wereexamined for external, visceral, and skeletal malformations.Although DCB was toxic to the dams at 64 mg/ kg/day, developmentaltoxicity was not detected. 3,4-TCB administration was followedby a significant (p < 0.01) increase in the average percentageof malformed fetuses per litter at 4 (7.2%), 8 (9.8%), 16 (25.4%),32 (50.0%), and 64 (75.0%) mg/kg/day versus the vehicle controlgroup (1.1%). None of the dosages tested was lethal to any ofthe dams. Significant decreases in maternal weight gain wereobserved at 16 mg/kg/day and above; however, the differencesfrom the control value most likely were due to significant decreasesin the mean number of live fetuses per dam, as the result ofreductions in the number of implants per dam, and significantincreases in the incidence of resorptions. Vaginal bleedingand other evidence of abortifacient effects also were presentin several dams in groups receiving 3,4-TCB at 16 mg/kg/dayand above. Cleft palate and hydronephrosis (significantly increasedat dosages of 4 mg/kg/day and above) were the predominant malformationsdetected. Thus, 3,4-TCB was found to be toxic to the conceptusat dosages of 4 mg/kg/day and above. Neither 3,5-TCB nor TMBshowed indications of maternal or developmental toxicity at64 mg/kg/day.     

Failure to induce mutations in Chinese hamster V79 cells and WB rat liver cells by the polybrominated biphenyls, Firemaster BP-6, 2,2',4,4',5,5'-hexabromobiphenyl, 3,3',4,4',5,5'-hexabromobiphenyl, and 3,3',4,4'-tetrabromobiphenyl

Firemaster BP-6 (FM), a mixture of polybrominated biphenyls (PBB), and the congeners 2,2',4,4',5,5'-hexabromobiphenyl (2,4,5-HBB), 3,3',4,4',5,5'-hexabromobiphenyl (3,4,5-HBB), and 3,3',4,4'-tetrabromobiphenyl (3,4-TBB) were tested for their ability to induce mutations in mammalian cells in culture. A rat liver microsome-mediated (S 15) Chinese hamster V79 cell mutation assay was used to test the mutagenicity of PBB and 3,4-TBB. V79 cells and WB rat liver cells were used to detect the mutagenicity of 2,4,5-HBB and 3,4,5-HBB. No mutagenic effects were detected at the dose levels tested. The possibility that these compounds promote liver neoplasms via a nongenotoxic mechanism is discussed.     

Metabolism of 2,2',3,3',6,6'-hexachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl by human hepatic microsomes

Since the metabolism of polychlorinated biphenyls (PCBs) is the critical factor that determines whether or not they accumulate in adipose tissue, we have studied the metabolism of two hexachlorobiphenyls (HCBs), 2,2'3,3',6,6'-hexachlorobiphenyl (236-HCB) and 2,2'4,4',5,5'-hexachlorobiphenyl (245-HCB), by human hepatic microsomes. Human microsomes were isolated from patients undergoing liver resection and were found to have cytochrome P-450 levels (0.28 nmoles/mg microsomal protein) and cytochrome P-450-dependent enzymatic activities similar to those reported by other workers. 245-HCB was not metabolized by human microsomes under various conditions, while 236-HCB was metabolized with an apparent Km of 8.8 microM and a Vmax of 5.1 pmoles/mg microsomal protein/min. Two major metabolites were formed and identified by gas chromatography-mass spectrometry as 2,2',3,3',6,6'-hexachloro-4-biphenylol and 2,2',3,3'6,6'-hexachloro-5-biphenylol. [14C]236-HCB equivalents were found to covalently bind to microsomal protein. Addition of 1 or 5 mM reduced glutathione decreased the degree of covalent binding. These data suggest that HCBs are metabolized through an arene oxide. The fact that 245-HCB was not metabolized explains why it is the predominant PCB found in human adipose tissue.     

Opposite Effects of 2,2',4,4',5,5'-Hexachlorobiphenyl and 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Antibody Response to Sheep Erythrocytes in Mice

The effect that cotreatment with 2,2',4,4',5,5'-hexachlorobiphe-nyl(PCB153) and 2,3,7,8-terrachlorodibenzo-p-dioxin (TCDD) hason the antibody plaque-forming cell (PFC) response to sheepred blood cells (SRBCs) was determined in female B6C3F1 mice.Groups of eight mice per group were given a single oral doseof PCB153 alone (0, 3.58, 35.8, or 358 mg/kg), TCDD alone (0,0.1, 1, or 10 µg/kg), and all possible combinations ofthese doses in corn oil 7 days prior to immunization with SRBCs.Separate groups of mice were given phenobarbital (PB) parenterallyby intraperito-neal injection at a dosage of 160 mg/kg/day for3 days. Four days after intravenous immunization, body, spleen,thymus, and liver weights and the PFC response to SRBCs weredetermined. Exposure to TCDD alone resulted in a dose-relatedsuppression of the PFC response, with significant suppressionat 1 and 10 µg/kg. In contrast, exposure to PCB153 aloneresulted in the enhancement of the PFC response at 358 mg/kg.Combined exposure to 358 mg/ kg PCB153 and TCDD resulted inno change (PCB153 + 0.1 µg/ kg TCDD) or suppression (PCB153+ 1 or 10 µg/kg TCDD) of the PFC response relative toPCB153 alone; however, the PFC response was enhanced (PCB153+ 0.1 µ/kg TCDD), unaffected (PCB153 + 1 µ/kg TCDD),or suppressed (PCB153 + 10 µ/kg TCDD) relative to cornoil controls. PB did not affect the PFC response to SRBCs, despitea 13-fold induction of hepatic pentoxyresorufin O-dealkylase(PROD) activity. These results suggest that PCB153 enhancementof the PFC response is not related to PROD induction and thatit acts as a functional antagonist rather than an aryl hydrocarbonreceptor or dispositional antagonist. By enhancing the PFC responseto SRBCs, PCB153 raises the "setpoint" response level. Consequently,cotreatment with an immunosuppressive dose of TCDD fails tosuppress the PFC response relative to corn oil controls, whileclearly suppressing it relative to the appropriate control,PCB153 alone.     

Acute pathologic effects of 3,3',4,4',5,5'-hexabromobiphenyl in rats: comparison of its effects with Firemaster BP-6 and 2,2',4,4',5,5'-hexabromobiphenyl

Male Sprague-Dawley rats were fed diets containing 0, 0.1, 1, 10, or 100 ppm of 3,3′,4,4′,5,5′-hexabromobiphenyl (HBB), 2,2′,4,4′,5,5′-HBB, or Firemaster (FM) BP-6, a commercial mixture of polybrominated biphenyls, for 9 days. Although 3,3′,4,4′,5,5′-HBB is not in FM BP-6, it was used because it is a 3-methylcholanthrene (MC)-type inducer of hepatic drug-metabolizing enzymes. Nearly one-half of FM BP-6 is comprised of 2,2′,4,4′,5,5′-HBB and this congener is strictly a phenobarbital (PB)-type inducer. FM BP-6 has both MC- and PB-type induction capability. Feed consumption and body and organ weights were recorded and histologic and ultrastructural changes were evaluated. Significant effects on feed intake and body weight occurred only at 10 or 100 ppm of 3,3′,4,4′,5,5′-HBB. Therefore, two of six rats given 100 ppm of 3,3′,4,4′,5,5′-HBB were continued on the diet until death occurred at 20 days. Liver weights were increased by each of the three chemicals at 10 and 100 ppm. Hepatocytes were diffusely enlarged and contained lipid vacuoles. The degree of vacuolation was dose related, most prominent in the centrolobular to midzonal area, and most severe in rats given 3,3′,4,4′,5,5′-HBB. Thymic and splenic weights were decreased at 10 and 100 ppm of 3,3′,4,4′,5,5′-HBB and lymphocytic depletion was severe in the thymus. Ultrastructural hepatic lesions were seen with all three chemicals. For 2,2′,4,4′,5,5′-HBB and FM BP-6 at 10 and 100 ppm the changes consisted mainly of increased smooth endoplasmic reticulum and lipid vacuolation. Additional changes seen with 3,3′,4,4′,5,5′-HBB included disorganization of rough endoplasmic reticulum, myelin body formation, and bile ductule hyperplasia. Results indicated that 3,3′,4,4′,5,5′-HBB causes more severe pathologic effects than either FM BP-6 or 2,2′,4,4′,5,5′-HBB.     

Interactive Effects between 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,2',4,4',5,5'-Hexachlorobiphenyl in Female B6C3F1 Mice: Tissue Distribution and Tissue-Specific Enzyme Induction

The distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) was studiedin female B6C3F1 mice. Single doses of TCDD alone (0, 0.1, 1,or 10 µg [³H]TCDD/kg), PCB 153 alone (0, 3.58, 35.8, or358 mg [¹⁴C]PCB 153/kg), and all possible combinations of thesedoses were administered in corn oil, po. At 7 days after dosing,11 different tissues were analyzed for radioactivity. When TCDDwas administered alone, TCDD-derived radioactivity distributedto all tissues in a dose-dependent manner, increasing with dosein the liver, while decreasing (as a percentage of the administereddose) in all other tissues. When PCB 153 was administered alone,the PCB 153 concentration was dose-dependently (percentage ofdose) decreased in liver, skin, lung, adrenals, kidney, andblood; no dosimetric effects were observed in the other organs.Coadminis-tration of low doses of both TCDD and PCB 153 resultedin little or no effect on the distribution of either compound.Interactive effects occurred in the pharmacokinetic behaviorof both compounds only at higher doses. For example, the amountof TCDD in the liver was increased by 358 mg PCB 153/kg. Inmost other organs administration of PCB 153 resulted in a dose-dependentdecrease in the TCDD content Coadministration of PCB 153 with10 µg TCDD/kg increased PCB 153 in the liver, but notin other tissues. These results clearly demonstrate that interactiveeffects on pharmacokinetic behavior occur only at high doses.     

Induction of altered hepatic foci by a mixture of dioxin-like compounds with and without 2,2',4,4',5,5'-hexachlorobiphenyl in female Sprague-Dawley rats

The hepatic tumor-promoting activity of a mixture of polyhalogenated aromatic hydrocarbons (PHAHs) was studied in a medium term two-stage initiation/promotion bioassay in female Sprague-Dawley rats. The PHAH mixture contained 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1, 2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,4,7, 8-pentachlorodibenzofuran (PeCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,3',4,4',5-pentachlorobiphenyl (PCB 118), 2,3,3',4,4', 5-hexachlorobiphenyl (PCB 156), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) and covered >90% of the total toxic equivalents (TEQ) present in Baltic herring. To determine possible interactive effects of di-ortho-substituted PCBs, the PHAH mixture was tested with (PHAH+) and without (PHAH-) PCB 153. Rats were initiated by a diethylnitrosamine injection (30 mg/kg body wt i.p.) 24 h after a partial 23 hepatectomy. Six weeks after initiation, the PHAH mixtures were administered once a week by subcutaneous injections for 20 weeks. Treatment with the PHAH mixtures caused liver enlargement and an increased activity of the hepatic cytochrome P4501A1/2 and P4502B1/2. All PHAH exposure groups exhibited an increased occurrence of hepatic foci positive for the placental form of glutathione-S-transferase. In the PHAH-group dosed 1 microgram TEQ/kg body wt/week, the volume fraction of the liver occupied by foci was significantly lower compared to the TEQ equivalent dosed TCDD group (3.8 vs 8.7%). The volume fraction was significantly increased in the groups treated with 0.5, 1, or 2 micrograms TEQ/kg body wt/week of the PHAH+ mixture (4.5, 5.2, and 6.6%, respectively) compared to the corn oil group (2.0%), but to a lower extent than expected on basis of the TEQ doses. Overall, the TEQ-based administered dose overestimated the observed tumor-promoting effects of this PHAH mixture. The applicability of the toxic equivalency factor concept, the role of differences in toxicokinetic properties and interactive effects of PCB 153 on hepatic deposition of the dioxin-like congeners are discussed.     

Neonatal exposure to the brominated flame-retardant, 2,2',4,4',5-pentabromodiphenyl ether, decreases cholinergic nicotinic receptors in hippocampus and affects spontaneous behaviour in the adult mouse

Polybrominated diphenyl ethers (PBDEs) are used as flame-retardants and have recently been shown to increase in the environment and in human milk. We have recently reported that neonatal exposure to 2,2',4,4',5-pentaBDE (PBDE 99) can induce persistent aberrations in spontaneous behaviour and also affect learning and memory functions in the adult animal. The present study indicates that the cholinergic system, in its developing stage, can be a target for PBDEs. Neonatal oral exposure of male NMRI mice, on postnatal day 10, to 2,2',4,4',5-pentaBDE (12mg/kg body weight) was shown to decrease the density of cholinergic nicotinic receptors in hippocampus, at an adult age. These findings show similarities to observations made from neonatal exposure to 2,2',4,4',5,5'-hexaBDE, PCBs and nicotine, compounds shown to affect cholinergic nicotinic receptors. The animals of the present study also showed disrupted spontaneous behaviour, and the highest dose that did not cause any significant behavioural disruption was 0.4mg 2,2',4,4',5-pentaBDE/kg b.w.     

Toxicity of 3,3',4,4'- and 2,2',5,5'-tetrabromobiphenyl: correlation of activity with aryl hydrocarbon hydroxylase induction and lack of protection by antioxidants

3,3',4,4'-Tetrabromobiphenyl is a minor component of commercial polybrominated biphenyl (PBB) mixture fireMaster BP-6 and is a potent inducer of aryl hydrocarbon hydroxylase (AHH). A single ip dose of 3,3',4,4'-tetrabromobiphenyl (150 mumol/kg) caused significant reduction in the growth rate in the immature male Wistar rat, as well as pale enlarged livers and marked reduction in thymus size. Under light microscopy, hepatocytes were enlarged and vacuolated. The vacuoles, which were most prominent in the midzonal region of the lobule, corresponded to fat droplets in oil-red-O-stained sections. The thymus, especially the cortex, was markedly depleted of lymphocytes. Neither the reduced growth, altered organ weights nor the histopathology was reversed for the duration of the study by the coadministration of the antioxidants butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), or vitamin E. Vitamin E did, however counter the negative effect of 3,3',4,4'-tetrabromobiphenyl on growth during the first 5 d of the study. 2,2',5,5'-Tetrabromobiphenyl, also a minor component of fireMaster BP-6, is a weak phenobarbital-type inducer of cytochrome P-450. When administered at the same dose, 2,2',5,5'-tetrabromobiphenyl did not elicit any observed toxic effects. These data confirm the correlation between AHH induction and toxicity for these PBBs and suggest that 3,3',4,4'-tetrabromobiphenyl may significantly contribute to the toxicity of fireMaster BP-6. Although there is evidence that polychlorinated biphenyls, and perhaps 2,3,7,8-tetrachlorodibenzo-p-dioxin, exert certain toxic effects via a lipid peroxidation mechanism, the toxic changes measured during this study were not reversed by the administration of the antioxidants.     

Polychlorinated and polybrominated biphenyl congeners and retinoid levels in rat tissues: structure-activity relationships.

The purpose of this study was to examine the structural requirements of polychlorinated and polybrominated biphenyls (PCBs and PBBs) for altering tissue levels of retinoids. Seven congeneric PCBs and PBBs were studied: 3,3',4,4'-tetrachlorobiphenyl (TCB), 2',3,3',4,5- and 3,3',4,4',5-pentachlorobiphenyls (-PeCBs), 3,3',4,4'- and 3,3',5,5'-tetrabromobiphenyls (-TBBs), 2,2',3,3',5,5'-hexachlorobiphenyl (-HCB), and 3,3',4,4',5,5'-hexabromobiphenyl (-HBB). Male Sprague-Dawley rats were fed a vitamin A-adequate diet (1.3 mg/kg) for 30 days before being given a single IP injection of one of seven polyhalogenated biphenyls (150 mumol/kg) in corn oil (10 ml/kg) or vehicle alone. Rats were killed 1 week later. Except for 3,3',4,4',5,5'-HBB, all PCBs and PBBs studied significantly decreased serum retinol levels and, except for 3,3',4,4',5,5'-HBB and 2,2',3,3',5,5'-HCB, all PCBs and PBBs also lowered the serum retinol-binding-protein (RBP) content. The activity of hepatic retinyl ester hydrolase (REH) was reduced by the treatment of 3,3',4,4',5-PeCB, 3,3',4,4'-TBB, and 3,3',4,4',5,5'-HBB. The levels of hepatic retinol were decreased by 2,2',3,3',5,5'-HCB, 2',3,3',4,5-PeCB, and 3,3',4,4',5-PeCB, while levels of hepatic retinyl palmitate were decreased by 2',3,3',4,5-PeCB, 3,3',4,4',5-PeCB, 3,3',4,4'-TCB, 3,3',4,4'-TBB, and 3,3',4,4',5,5'-HBB. The substantial decreases in hepatic retinyl palmitate levels could not be explained solely on the basis of hepatomegaly caused by acutely toxic PCBs and PBBs. All halogenated biphenyls which caused a decrease in hepatic retinyl palmitate also caused an increase in renal retinyl palmitate except 3,3',4,4',5-PeCB. In summary, the acutely toxic (nonortho substituted) congeners had pronounced effects on hepatic, renal, and serum retinoids whereas other biphenyls only decreased serum retinol levels. The effects of these seven compounds on REH activity were not correlated with the effects on serum retinol or RBP levels. Therefore, this study shows that the structure-activity relationships for altering hepatic retinoids differ from those for serum retinol, implying the involvement of multiple mechanisms.     

Subchronic Toxicity of 3,3',4,4',5-Pentachlorobiphenyl in the Rat: I. Clinical, Biochemical, Hematological, and Histopathological Changes

The systemic, toxicity of 3,3',4,4',5-pentachlorobiphenyl (PCB126) following subchronic dietary exposure was investigatedin Sprague-Dawley rats. PCB 126 was administered to rats ofboth sexes at concentrations of 0.1, 1.0, 10, or 100 ppb intheir diet for 13 weeks. Another group of rats received a loadingdose of 5 µg PCB/kg body wt at the start of the feedingperiod followed by exposure to 10 ppb PCB diet for the sameperiod of time as the other groups. Growth suppression and decreasedfood consumption were observed in the highest dose groups ofboth sexes. Increased organ/body weight ratios for the liveroccurred in the 10 and 100 ppb groups of both sexes. Rats ofboth sexes exposed to the highest dose of the PCB also exhibitedincreased relative kidney, spleen, and brain weights. Hematologicaland most serum biochemical changes were confined to the 100ppb groups. These included elevated alkaline phosphatase, bilirubin,cholesterol, and aspartate aminotransferase, and decreased serumglucose, hemoglobin, erythrocytes, hematocrit, and platelets.A dose-dependent increase in liver ethoxyre-sorufin-O-deethylaseactivity was observed in rats of both sexes starting at 0.1ppb. A dose-dependent increase in liver uroporphyrin levelswas observed in both sexes and significant changes occurredin the female rats at 1.0 ppb and higher dose groups. Decreasedliver vitamin A was observed in the 10 ppb group and higherin both sexes. Kidney vitamin A was elevated in the 100 ppbgroup. No statistically significant changes were noted in concentrationsof brain biogenic amines. PCB 126 residues were 10-fold higherin liver than in fat. Treatment-related histopathological changeswere observed in the thymus, thyroid, bone marrow, and liverof rats exposed to the 10 ppb diet, but increased frequencyof mild changes was observed in most of these tissues at the1.0 ppb level. Based on the above data, the no adverse effectlevel was judged to be 0.1 ppb in the diet or 0.01 µg/kgbody wt/day.     

The pharmacokinetics of 2,2',5,5'-tetrachlorobiphenyl and 3,3',4,4'-tetrachlorobiphenyl and its relationship to toxicity

The pharmacokinetics of two toxicologically diverse tetrachlorobiphenyls (TCBs) were measured in mice. After dosing to apparent steady-state conditions, 2,2',5,5'-TCB was found to have a tissue elimination half-life of between 1.64 and 2.90 days. The half-life of 3,3',4,4'-TCB was similar, ranging from 1.07 to 2.60 days. Systemic clearance and volume of distribution estimates were also similar for the two TCB isomers. The 3,3',4,4'-isomer had a substantially greater partitioning from serum into adipose, liver, and thymic tissues. With dosing regimens developed using these measured pharmacokinetic parameters, experiments were undertaken to compare toxic potency of these two TCBs when similar tissue concentrations of the two isomers were achieved in target and storage tissues. These studies demonstrated that thymic atrophy occurs at lower 3,3',4,4'-TCB doses and tissue concentrations than those required to produce hepatotoxicity. These two organ toxicities were produced only by 3,3',4,4'-TCB despite the fact that equivalent or higher tissue concentrations of 2,2',5,5'-TCB were achieved in vivo in all tissues. We conclude that the in vivo difference in the toxic potency of these two TCB isomers does not result from the significant differences in their tissue disposition, elimination, and ultimate bioaccumulation.     

Immunosuppressive and Monooxygenase Induction Activities of Highly Chlorinated Diphenyl Ether Congeners in C57BL/6 and DBA/2 Mice

The dose-response effects of 2,2',3,3',4,5,5',6,6'–,2,2',3,3',4,4',5,6,6'-and2,2',3,3',4,4',5,5',6-nonachlorodiphenyl ether (non-aCDE) anddecachlorodiphenyl ether (decaCDE) on the splenic plaque-formingcell (PFC) response to sheep red blood cells (SRBCs) and theinduction of hepatic microsomal ethoxyresorufin O-deethylase(EROD) activity was determined in aryl hydrocarbon (Ah)-responsiveC57BL/6 and less Ah-responsive DBA/2 mice. All the congenersexhibited immunotoxicity at doses between 2.5 and 10 µmol/kgin C57BL/6 mice whereas in DBA/2 mice doses25 µmol/kgwere required to cause inhibition of the PFC response to SRBCs.The results also showed that the nonaCDE isomers and decaCDEwere more active as inducers of hepatic EROD activity in C57BL/6than DBA/2 mice; however, there was not a correlation betweenthe induced EROD activity and the CYP1A1 and CYP1A2 mRNA levelsin the C57BL/6 mice. These data suggested that the immunotoxicityof these compounds was mediated through the Ah receptor. However,the results showed that the immunotoxicity of the nonaCDE isomersand decaCDE was unexpectedly high compared to that of lowerchlorinated diphenyl ethers and there were no apparent structure-activityrelationships among the higher chlorinated congeners. This suggeststhat some of the immunosuppressive effects observed for thenonaCDE isomers and decaCDE may be Ah receptor-independent.     

Immunosuppressive and monooxygenase induction activities of polychlorinated diphenyl ether congeners in C57BL/6N mice: quantitative structure-activity relationships

The dose-response effects of several polychlorinated diphenyl ether (polyCDE) congeners on the inhibition of the splenic plaque-forming cell (PFC) response to sheep red blood cell antigen and the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities were determined in male C57BL/6 mice. The immunotoxic potencies for the polyCDE congeners (ED50 values for the suppression of PFCs/spleen and PFCs/10(6) cells) followed the order 2,3,3',4,4',5-hexaCDE (0.5 and 2.2 mumols/kg) greater than 3,3',4,4',5-pentaCDE (8.8 and 5.1 mumols/kg) greater than 2,3',4,4',5-pentaCDE (21.8 and 14.2 mumols/kg) greater than 3,3',4,4'-tetraCDE (50.6 and 28.7 mumols/kg) greater than 2,2',4,4',5,5'-hexaCDE (81.2 and 56.5 mumols/kg) greater than 2,2',4,5,5'-pentaCDE (258 and 228 mumols/kg) greater than 2,2',4,4',5,6'-hexaCDE (greater than 400 mumols/kg for both responses). The potencies of the polyCDE congeners as inducers of hepatic microsomal AHH and EROD activities were similar to their immunotoxicities and only one compound, namely, 2,3',4,4',5,5'-hexaCDE, did not cause dose-response immunosuppressive effects in the mice. The structure-activity relationships for the polyCDEs exhibited both differences and similarities. For example, the coplanar 3,3',4,4'-tetraCDE and 3,3',4,4',5-pentaCDE congeners were less immunotoxic than their monoortho 2,3',4,4',5-pentaCDE and 2,3,3',4,4',5-hexaCDE analogs, respectively, and similar results were also observed for their enzyme induction potencies. For the corresponding polychlorinated biphenyls (PCB) congeners the coplanar compounds were significantly more active than their monoortho analogs. In addition, two diortho-substituted compounds, namely, 2,2',4,5,5'-pentaCDE and 2,2',4,4',5,5'-hexaCDE, were also immunotoxic at a dose of 400 mumols/kg whereas, their PCB analogs were inactive. These studies clearly demonstrate that for the polyCDE congeners, increasing ortho-chloro substitution is less effective in reducing the activity of these congeners compared to the well-recognized ortho effects reported for the PCBs. The differences in the structure-activity relationships between polyCDEs and PCBs are related to the ether bridge in the polyCDEs in which the resultant increased bond length between the two phenyl rings thereby diminishes the effects of ortho substituents on the biochemical and toxic potencies of these compounds.     

Promotion of Altered Hepatic Foci by 2,3',4,4',5-Pentachlorobiphenyl in Sprague-Dawley Female Rats

The tumor promotion potential of 2,3',4,4',5-pentachlorobiphenyl(PCB-118) was studied in a two-stage initiation/promotion bioassayin female Sprague-Dawley rats. The animals were initiated byintraperitoneal administration of N-nitrosodiethylamine afterpartial hepatectomy. After 5 weeks of recovery, the promotionperiod commenced by once-weekly subcutaneous administrationsof PCB-118 at six dose levels (10, 40, 160, 640, 2500, and 10,000µg/kg body weight/week) for 20 weeks. In addition, threeof these dose levels (40, 640, and 10,000 µg/kg body weight/week)were administered for 52 weeks. Evaluation of hepatic foci positivefor glutathione S-transferase P demonstrated that the mono-orthochlorine substituted congener PCB-118 significantly increasedthe number of foci/cm³ of liver in the two highest dose groupsafter 20 weeks, but did not significantly increase the percentageof the liver occupied by foci. After 52 weeks of treatment,both the percentage and the number of foci/cm³ were significantlyincreased in the highest dose group. A toxic equivalency factorbased on foci development during 20 weeks of treatment wouldbe less than 0.00002. Altered relative liver and thymus weightswere observed after treatment with both substances as well asan induction of methyl cholanthrene- and phenobarbital-inducibleisoenzymes of cytochrome P450 monooxygenase. These results showthat PCB-118 has a potency to enhance foci growth in rat liver,although the potency is low compared to that of structurallyrelated compounds.     

Developmental Toxicity of PCB 126 (3,3',4,4',5-Pentachlorobiphenyl) in Nestling American Kestrels (Falco sparverius)

Planar PCB congeners are embryotoxic and teratogenic to birdsincluding American kestrels. The developmental toxicity of 3,3',4,4',5-pentachlorobiphenyl(PCB 126) was studied in the posthatching kestrel as a modelfor the eagle. Nestlings were dosed orally for 10 days with5 µl/g body weight of corn oil (controls) or the planarPCB 126 at concentrations of 50, 250, or 1000 ng/g body weight.Dosing with 50 ng/g of PCB 126 resulted in a hepatic concentrationof 156 ng/g wet weight, liver enlargement and mild coagulativenecrosis, over 10-fold increases in hepatic microsomal ethoxyresorufin-O-dealkylaseand benzyloxyresorufin-O-dealkylase, and approximately a 5-foldincrease in methoxyresorufin-O-dealkylase. At this dose, mildto moderate lymphoid depletion of the spleen was apparent, aswere decreased follicle size and content of the thyroid. At250 ng/g, concentration of PCB 126 in the liver was 380 ng/gwith increasing multifocal coagulative necrosis, decreased bonegrowth, decreased spleen weight with lymphocyte depletion ofthe spleen and bursa, and degenerative lesions of the thyroid.At 1000 ng/g, the liver concentration was 1098 ng/g, accompaniedby decreased bursa weight, decreased hepatic thiol concentration,and increased plasma enzyme activities (ALT, AST, and LDH-L)in addition to the previous effects. Highly significant positivecorrelations were noted between liver concentrations of PCB126 and the ratio of oxidized to reduced glutathone. These findingsindicate that nestling kestrels are more susceptible to PCB126 toxicity than adults, but less sensitive than embryos, andthat planar PCBs are of potential hazard to nestling birds.     

Effects of pure chlorobiphenyls (2,4',5-trichlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl) on the reproductive capacity in female mice.

The effects of two pure chlorobiphenyls (2,4',5-trichlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl) on the reproductive capacity in female mice have been studied. The results obtained show that females given 0.5 mg 2,5',5-trichlorobiphenyl (TCB)/day or 0.5 mg 2,2',4,4',5,5'-hexachlorobiphenyl (HCB)/day for six consecutive days, starting on the first day of pregnancy, have significantly lower frequencies of implanted ova than the control females, whereas the frequency of implanted ova does not differ either between females given 0.05 mg/day of TCB or HCB and the control females, or between females given TCT 0.05 mg/day or 0.5 mg/day and those given the corresponding dose of HCB. Females given 0.5 mg/day of TCB or HCB, respectively, also have significantly higher contents in the livers of cytochrome P-450, on Day four of pregnancy, than the control females. The results also show that, under the experimental conditions used, neither the frequency of pregnancies nor the number of implanted ova per pregnant female differ significantly between the different groups.