In Uteroand Lactational Exposure to PCBs in Mice: Adult Offspring Show Altered Learning and Memory Depending onCyp1a2andAhrGenotypes

Polychlorinated biphenyls (PCBs) are persistent toxic pollutants occurring as complex mixtures in the environment. Humans are known genetically to have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2) levels and > 12-fold differences in aryl hydrocarbon receptor (AHR) affinity, both of which could affect PCB pharmacokinetics. Thus, we compared Ahr(b1)_Cyp1a2(+/+) high-affinity AHR wild-type, Ahr(d)_Cyp1a2(+/+) poor affinity AHR wild-type, Ahr(b1)_Cyp1a2(-/-) knockout, and Ahr(d)_Cyp1a2(-/-) knockout mouse lines. We chose a mixture of three coplanar and five noncoplanar PCBs to reproduce that seen in human tissues, breast milk, and the food supply. The mixture was given by gavage to the mother on gestational day 10.5 (GD10.5) and postnatal day 5 (PND5); tissues were collected from pups and mothers at GD11.5, GD18.5, PND6, PND13, and PND28. Ahr(b1)_Cyp1a2(-/-) pups showed lower weight at birth and slower rate of growth postnatally. Absence of CYP1A2 resulted in significant splenic atrophy at PND13 and PND28. Presence of high-affinity AHR enhanced thymic atrophy and liver hypertrophy in the pups. Concentrations of each congener were analyzed at all time points: maximal noncoplanar congener levels in maternal tissues were observed from GD18 until PND6, whereas the highest levels in pups were found between PND6 and PND28. Coplanar PCB concentrations were generally higher in Ahr(d)-containing pup tissues; these findings are consistent with earlier studies demonstrating the crucial importance of AHR-mediated inducible CYP1 in the gastrointestinal tract as a means of detoxication of oral planar polycyclic aromatic hydrocarbons.

Polychlorinated biphenyls (PCBs) have been shown to be embryotoxic. The mechanism(s) of action is not clearly understood. The toxic effects could be either direct or indirect. Furthermore, PCB congeners vary in their toxic potential. They can be classified in coplanar PCBs binding to the transcription factor aryl hydrocarbon receptor (AhR), which induce subsequent changes in gene expression, and noncoplanar PCBs exhibiting AhR-independent effects. In order to investigate possible mechanisms, 5 and 6 days old preimplantation rabbit embryos were exposed in vitro to low levels of coplanar (PCB 77, 126, and 169) or noncoplanar PCBs (PCB 28, 52, 101, 118, 138, 153, and 180). The PCB effects were studied by semiquantitative RT-PCR analysis of AhR target genes (cytochrome P450 (CYP) 1A1, 1A2, UDP-glucuronosyl transferase 1, glutathione S-transferase pi1 and aldehyde dehydrogenase) and dioxin-responsive genes (IL 1beta, PAI 2, Cox 2, TGFalpha, EGF, erbB 1-4, c-fos, c-jun, HSP 90, cyclophilin 40), and by differential display (DD) RT-PCR. CYP 1B1 mRNA and AhR protein were localized by in situ hybridization and immunohistochemistry, respectively. From the AhR target genes studied only CYP 1B1, and cyclooxygenase 2 showed an increase in mRNA levels after coplanar and noncoplanar PCB. Interleukin 1beta and plasminogen activator inhibitor 2 were downregulated. CYP 1B1 mRNA showed a stage specific inducibility at day 6, but not at day 5. By DD RT-PCR we identified six new genes previously not reported to be regulated by PCBs. The mRNAs encoding the subunits 1, 2, 4, and 5 of the NADH ubiquinone oxidoreductase and beta-globin showed a decrease, whereas trichohyalin mRNA was increased after PCB exposure. Coplanar and noncoplanar PCB congeners elicited similar responses on the mRNA levels of the studied genes. Exposure to coplanar PCBs did not result in the AhR being translocated to the nucleus. Our results show that (i). PCBs induce changes in gene expression in rabbit day 5 and 6 preimplantation embryos and imply (ii). that the transcriptional changes observed were not mediated by the nuclear AhR.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolk AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (˜2–7 fmol/mg protein) were much lower than observed generally in rodents (˜50–300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59–60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducibk TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorod-ibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A 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 competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat The Km for ethoxyresonifin (0.095 μM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresonifin than that of rat CYP1A.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A 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 competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.

AhrdCyp1a2(−/−) mice show increased susceptibility to PCB-induced developmental neurotoxicity

Exploratory ab initio MO calculations on the structures of polychlorinated biphenyls(PCBs): a possible way to make a coplanar PCB stable at coplanar conformation

Non-coplanar and coplanar polychlorinated biphenyls potentiate genotoxicity of aflatoxin B1 in a human hepatocyte line by enhancing CYP1A2 and CYP3A4 expression

Polychlorinated biphenyls (PCBs) are accumulated in our body through food chain and cause a variety of adverse health effects including neurotoxicities such as cognitive deficits and motor dysfunction. In particular, neonates are considered as a high risk group for the neurotoxicity of PCBs exposure. The present study attempted to analyze the structure-activity relationship among PCB congeners and the mechanism of PCBs-induced neurotoxicity. We measured total protein kinase C (PKC) activities, PKC isoforms, reactive oxygen species (ROS), and induction of neurogranin (RC-3) and growth associated protein-43 (GAP-43) mRNA in cerebellar granule cells of neonatal rats with phorbol 12, 13-dibutyrate ([3H]PDBu) binding assay, western blot, ROS assay, and reverse transcription PCR (RT-PCR) analysis respectively following the different structural PCBs exposure. Only non-coplanar PCBs showed a significant increase of total PKC-α and βII activity as measured with [3H]PDBu binding assay. ROS were more increased with non-coplanar PCBs than coplanar PCBs. The mRNA levels of RC-3 and GAP-43 were more induced with non-coplanar PCBs than coplanar PCBs, indicating that these factors may be useful biomarkers for differentiating non-coplanar PCBs from coplanar PCBs. Non-coplanar PCBs may be more potent neurotoxic congeners than coplanar PCBs. This study provides evidences that non-coplanar PCBs, which have been neglected in the risk assessment processes, should be added in the future to improve the quality and accuracy of risk assessment on the neuroendocrinal adverse effects of PCBs exposures.

Comparison of hepatic and extra hepatic induction of cytochrome P4501A by graded doses of aryl hydrocarbon receptor agonists in Atlantic tomcod from two populations

Coplanar polychlorinated biphenyl-induced CYP1A1 is regulated through caveolae signaling in vascular endothelial cells

Proinflammatory Properties of Coplanar PCBs: In Vitro and in Vivo Evidence

Polychlorinated biphenyls (PCBs), especially the more coplanar PCBs, have been shown to induce oxidative stress, various transcription factors, and subsequent inflammatory processes critical to atherosclerosis in vascular endothelial cells. Dietary flavonoids such as catechins and quercetin possess antioxidant and anti-inflammatory properties. To test the hypothesis that flavonoids can modify PCB-mediated endothelial cytotoxicity, endothelial cells were treated with epigallocatechin-3-gallate (EGCG; 5 to 50 muM) or quercetin (10 to 100 muM) with or without PCB 77 (3,3',4,4'-tetrachlorobiphenyl, 3.4 muM) for 6 h. EGCG and quercetin strongly, and in a concentration-dependent manner, inhibited oxidative stress induced by PCB 77 as measured by DCF fluorescence. The role of cytochrome P450 1A1 (CYP1A1) in the PCB-induced toxicity was investigated. EGCG at 50 muM and quercetin at 100 muM concentrations markedly inhibited CYP1A1 mRNA levels and enzyme activity. Furthermore, EGCG and quercetin downregulated the PCB 77-mediated increase in aryl hydrocarbon receptor (AhR)-DNA binding activity. These data suggest that protective effects of EGCG and quercetin are initiated upstream from CYP1A1 and that these flavonoids may be of value for inhibiting the toxic effects of PCBs on vascular endothelial cells.

The distribution of natural populations of American mink is restricted to locales that are in proximity to aquatic ecosystems. Because of the lipophilicity and persistence of polychlorinated biphenyls (PCBs) and reliance of mink on aquatic-based diets, mink at contaminated locales often bioacccumulate high levels of PCBs. In addition, in controlled laboratory studies, mink are highly sensitive at reproductive and developmental end points to the toxic effects of environmental PCB mixtures. It is believed that most, if not all, toxic effects of PCBs occur through activation of the aryl hydrocarbon receptor (AHR) pathway. Transcription of cytochrome P4501A1 (CYP1A1) by PCBs is also mediated through activation of AHR. Thus, levels of CYP1A1 mRNA provide a quantitative assay of exposure to and early biologic effect of PCBs on mink and may be predictive of toxicity at higher levels of biologic organization. We developed polymerase chain reaction (PCR) primers to amplify CYP1A1 as well as identified a housekeeping gene from mink cDNA. We used real-time reverse transcription-PCR to quantify and compare levels of hepatic CYP1A mRNA among groups of ranched mink kits and juveniles, which were fed diets or exposed in utero to fish that were low in PCBs (Atlantic herring) or to diets that were contaminated with three different levels of PCBs (carp) from Saginaw Bay, Lake Michigan. We found significant differences in CYP1A1 mRNA expression between mink fed the control diet and those fed a PCB-contaminated carp diet at all three treatment levels and exposure times. CYP1A1 mRNA was significantly induced 5.3- to 6.6-fold and 3.7- to 4.7-fold at 6 and 27 weeks, respectively. In previous studies, dietary exposures to PCB-contaminated carp were shown to cause mild to moderate lesions in the mandible and maxilla of these animals. This study demonstrates that hepatic CYP1A1 mRNA may be a sensitive biomarker of exposure of mink to environmentally relevant levels of PCBs and may be predictive of their effects in natural populations.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that remain a human health concern with newly discovered sources of contamination and ongoing bioaccumulation and biomagnification. Children exposed during early brain development are at highest risk of neurological deficits, but highly exposed adults reportedly have an increased risk of Parkinson's disease. Our previous studies found allelic differences in the aryl hydrocarbon receptor and cytochrome P450 1A2 (CYP1A2) affect sensitivity to developmental PCB exposure, resulting in cognitive deficits and motor dysfunction. High-affinity Ahr b Cyp1a2(-/-) mice were most sensitive compared with poor-affinity Ahr d Cyp1a2(-/-) and wild-type Ahr b Cyp1a2(+/+) mice. Our follow-up studies assessed biochemical, histological, and gene expression changes to identify the brain regions and pathways affected. We also measured PCB and metabolite levels in tissues to determine if genotype altered toxicokinetics. We found evidence of AHR-mediated toxicity with reduced thymus and spleen weights and significantly reduced thyroxine at P14 in PCB-exposed pups. In the brain, the greatest changes were seen in the cerebellum where a foliation defect was over-represented in Cyp1a2(-/-) mice. In contrast, we found no difference in tyrosine hydroxylase immunostaining in the striatum. Gene expression patterns varied across the three genotypes, but there was clear evidence of AHR activation. Distribution of parent PCB congeners also varied by genotype with strikingly high levels of PCB 77 in poor-affinity Ahr d Cyp1a2(-/-) while Ahr b Cyp1a2(+/+) mice effectively sequestered coplanar PCBs in the liver. Together, our data suggest that the AHR pathway plays a role in developmental PCB neurotoxicity, but we found little evidence that developmental exposure is a risk factor for Parkinson's disease.

Ahr and Cyp1a2 genotypes both affect susceptibility to motor deficits following gestational and lactational exposure to polychlorinated biphenyls