Richard F. Seegal

Lightly chlorinated ortho-substituted PCB congeners decrease dopamine in nonhuman primate brain and in tissue culture

Exposure of the nonhuman primate, Macaca nemestrina, to Aroclor 1016, a commercial mixture of 26 lightly chlorinated PCB congeners, decreased dopamine concentrations in the caudate, putamen, substantia nigra, and hypothalamus. Only three ortho-substituted nonplanar PCB congeners (2,4,4, 2,4,2,4, and 2,5,2,5) were detected in these brain regions, suggesting that these congeners may be responsible for the observed decreases in dopamine. The ability of these and other PCB congeners to alter dopamine function was tested directly by applying them to dopamine-synthesizing cells in culture, PC-12 pheochromocytoma cells. In vitro testing demonstrated that these three congeners reduced cellular dopamine concentrations while planar, dioxin-like congeners, e.g., 3,4,3,4 and 3,4,5,3,4, did not. Thus, these ortho-substituted nonplanar congeners may be directly responsible for the observed changes in in vivo neurochemistry. Furthermore, these results suggest that the observed decreases in both in vivo and in vitro dopamine concentrations may occur through a novel mechanism and not through the Ah-receptor complex thought to mediate immunotoxic and hepatotoxic changes following exposure to dioxin and dioxin-like PCBs.

Comparison of effects of Aroclors 1016 and 1260 on non-human primate catecholamine function

Adult male non-human primates, Macaca nemestrina, were orally-exposed to corn oil or corn oil containing either Aroclor 1016 or 1260 at doses of 0.8, 1.6 or 3.2 mg/(kg.day) for 20 weeks. Brain concentrations of biogenic amines and individual PCB congeners were determined following exposure. Aroclor 1016 significantly decreased concentrations of dopamine and its metabolites in the caudate, putamen, substantia nigra and hypothalamus but did not alter neurotransmitter or metabolite concentrations in the globus pallidus and hippocampus. Total PCB concentrations ranged from 1 to 5 ppm with only three congeners detected (2,4,4; 2,4,2,4 and 2,5,2,5) making up, on average, 72%, 18% and 7% respectively of the total residue in brain. There were no discernible differences in the congener make-up between brain regions. Aroclor 1260 reduced dopamine concentrations in the caudate, putamen and hypothalamus but produced no effects in the substantia nigra, globus pallidus or hippocampus. Aroclor 1260 concentrations ranged from 18 to 28 ppm with the highest levels found in the hippocampus. Of the congeners that made up more than 5% of the total residue in brain, all were hexa- and heptachlorinated di-ortho-substituted congeners. There were no discernible differences in congener make-up between brain regions. We conclude that: (1) ortho-substituted non-planar congeners are responsible for the observed changes in neurochemical function; (2) both Aroclor 1016 and Aroclor 1260 decrease dopamine concentrations by similar mechanisms; and (3) based on differences in brain concentrations of Aroclor 1260 congeners compared to Aroclor 1016 congeners, lightly-chlorinated congeners are more effective in reducing central dopamine concentrations than are the more highly chlorinated congeners.

Decreases in dopamine concentrations in adult, non-human primate brain persist following removal from polychlorinated biphenyls.

Adult male non-human primates, Macaca nemestrina, were orally exposed for 20 weeks to 3.2 mg/kg per day of either Aroclor 1016 or Aroclor 1260 made up in corn oil. Following cessation of exposure, the animals were observed for either an additional 24 or 44 weeks. After killing, regional brain concentrations of biogenic amines and polychlorinated biphenyls were determined by high-performance liquid chromatography (HPLC) and gas chromatography (GC), respectively. Brain dopamine (DA) concentrations were significantly decreased, compared to controls, in all polychlorinated biphenyl-exposed animals. Most importantly, in spite of significant decreases in brain polychlorinated biphenyl concentrations observed following removal from exposure (an average decline of 60%), there was no statistically discernible relationship of the changes in brain DA concentrations to either time following removal from polychlorinated biphenyls or brain polychlorinated biphenyl concentrations. These findings demonstrate that sub-chronic exposure of the adult non-human primate to polychlorinated biphenyls results in long-lasting changes in brain DA concentrations.

2,2',6,6'‐tetrachlorobiphenyl is estrogenic in vitro and in vivo

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants whose effects on biological systems depend on the number of and the positions of the chlorine substitutions. In the present study we examined the estrogenicity of the fully ortho‐substituted PCB, 2,2,6,6‐tetrachlorobiphenyl (2,2,6,6‐TeCB). This PCB was chosen as the prototypical ortho‐substituted PCB to test the hypothesis that ortho‐substitution of a PCB with no para‐ or meta‐chlorine‐substitutions results in enhanced estrogenic activity. The results indicate that 2,2,6,6‐TeCB is estrogenic both in vitro, in the MCF‐7 cell focus assay, and in vivo, in the rat uterotropic assay. The estrogenic activity elicited by the addition of 5 μM 2,2,6,6‐TeCB to the medium of MCF‐7 cultures was inhibited by the estrogen receptor (ER) antagonist, LY156758, suggesting that 2,2,6,6‐TeCB or a metabolite is acting through an ER‐dependent mechanism. Results from competitive binding assays using recombinant human (rh) ER indicate that 2,2,6,6‐TeCB does not bind rhERα or rhERβ. A metabolite of 2,2,6,6‐TeCB, 2,2,6,6‐tetrachloro‐4‐biphenylol (4‐OH‐2,6,2,6‐TCB), does bind rhERα and rhERβ and is also 10‐fold more estrogenic than 2,2,6,6‐TeCB in the MCF‐7 focus assay; however, this metabolite is not detected in the medium of MCF‐7 cultures exposed to 2,2,6,6‐TeCB. Taken together, the results suggest that the estrogenicity observed in human breast cancer cells and the rat uterus may be due to 1) an undetected metabolite of 2,2,6,6‐TeCB binding to the ER, 2) 2,2,6,6‐TeCB binding directly to a novel form of the ER, or 3) an unknown mechanism involving the ER. J. Cell. Biochem. 72:94–102, 1999.

Occupational exposure to PCBs reduces striatal dopamine transporter densities only in women: a β-CIT imaging study.

We hypothesize that occupational exposure to PCBs is associated with a reduction in central dopamine (DA) similar to changes previously seen in PCB exposed adult non-human primates. To test that hypothesis, we used [(123)I]beta-CIT SPECT imaging to estimate basal ganglia DA transporter density in former capacitor workers. Women, but not men, showed an inverse relationship between lipid-adjusted total serum PCB concentrations and DA transporter densities in the absence of differences in serum PCB concentrations. These sex differences may reflect age-related reductions in the levels of gonadal hormones since these hormones have been shown experimentally to alter response to DA neurotoxicants. These findings may aid in better understanding the roles that sex and age play in modifying central DA function following exposure, not only to PCBs, but also to other DA neurotoxicants as well as further elucidating the role of gonadal hormones in influencing the initiation and/or progression of neurodegenerative disorders.

Developmental Coexposure to Polychlorinated Biphenyls and Polybrominated Diphenyl Ethers Has Additive Effects on Circulating Thyroxine Levels in Rats

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are widespread environmental contaminants found in seafood and dairy products. PCBs and PBDEs are structurally similar chemicals and affect thyroid hormone function and behavior in children and laboratory rodents. Although coexposure frequently exists, the in vivo developmental effects of combined exposure to PCBs and PBDEs on thyroxine (T4) levels are unknown. We examined the effects of PCB and PBDE coexposure from gestational day 6 through postnatal day (p) 21, alone and in combination, on T4 levels in rat offspring. In males, exposure to PCBs and PBDEs at 1.7, 5, 10, 20, 40, and 60 μmol/kg/day induced equivalent and dose-dependent reductions in T4 from p 7 to p 21. Exposure to equimolar mixtures of PCBs and PBDEs at 3.4, 10, 20, 40, and 80 μmol/kg/day additively reduced T4 from p 7 to p 21 in males. In a second series of experiments, we determined sex effects on the mixture exposures and found that coexposure to PCBs and PBDEs had similar additive effects on T4 levels in male and female offspring. This study demonstrates that equimolar exposure to PCBs and PBDEs induces similar reductions in T4 levels and that coexposure to a mixture of PCBs and PBDEs has additive effects on T4 levels. These thyroid hormone effects of coexposure to PCBs and PBDEs are important when considering the cumulative effects of coexposure to multiple environmental thyroid hormone-disrupting agents in risk assessment for developmental disorders.

Polychlorinated Biphenyls and Polybrominated Diphenyl Ethers Alter Striatal Dopamine Neurochemistry in Synaptosomes from Developing Rats in an Additive Manner

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are widespread environmental contaminants associated with changes in behavior and neurochemical function in laboratory animals and behavioral deficits in children. PCBs and PBDEs are found in food, especially in seafood and dairy products, and coexposure to these contaminants is likely. We examined the effects of an environmentally relevant mixture of PCBs (Fox River Mix [FRM]) and a PBDE mixture (DE-71) alone and in combination on synaptosomal and medium dopamine (DA) levels and the levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in striatal synaptosomes derived from postnatal days (PND) 7, PND14, or PND21 rats. FRM elevated medium DA and reduced synaptosomal DA concentrations with greater potency than equimolar concentrations of DE-71. The effects of FRM, but not DE-71, were dependent on the age of the animals from which the synaptosomes were derived, with greater effects observed in synaptosomes from the youngest animals. We used Bliss model of independence to assess the possible interaction(s) of a 1:1 mixture of FRM and DE-71 on synaptosomal DA function and found that the effects of the FRM/DE-71 mixture were additive. Furthermore, as for FRM alone, the effects of the FRM/DE71 mixture were greater in synaptosomes prepared from PND7 rats than in synaptosomes from PND14 and PND21 rats. Because the effects of these contaminants are additive, it is necessary to take into account the cumulative exposure to organohalogen contaminants such as PCBs and PBDEs during risk assessment.

Polychlorinated biphenyl-induced oxidative stress in organotypic co-cultures: experimental dopamine depletion prevents reductions in GABA.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that have been demonstrated to be toxic to the dopamine (DA) systems of the central nervous system. One proposed mechanism for PCB-induced DA neurotoxicity is inhibition of the vesicular monoamine transporter (VMAT); such inhibition results in increased levels of unsequestered DA and DA metabolism leading to oxidative stress. We have used an organotypic co-culture system of developing rat striatum and ventral mesencephalon (VM) to determine whether alterations in the vesicular storage of DA, resulting from PCB exposure and consequent induction of oxidative stress, leads to GABA and DA neuronal dysfunction. Twenty-four-hour exposure to an environmentally relevant mixture of PCBs reduced tissue DA and GABA concentrations, increased medium levels of DA and measures of oxidative stress in both the striatum and VM. Alterations in neurochemistry and increases in measures of oxidative stress were blocked in the presence of n-acetylcysteine (NAC). Although NAC treatment did not alter PCB-induced changes in DA neurochemistry, it did protect against reductions in GABA concentration. To determine whether alterations in the vesicular storage of DA were responsible for PCB-induced oxidative stress and consequent reductions in GABA levels, we depleted DA from the co-cultures using alpha-methyl-p-tyrosine (AMPT). AMPT reduced striatal and VM DA levels by 90% and 70%, respectively. PCB exposure, following DA depletion, neither increased levels of oxidative stress nor resulted in GABA depletion. These results suggest that PCB-induced alterations in the vesicular storage of DA, resulting in increased levels of unsequestered DA, leads to increased oxidative stress, depletion of tissue glutathione, and consequent reductions in tissue GABA concentrations.

Dopaminergic development of prenatal ventral mesencephalon and striatum in organotypic co-cultures.

Using organotypic co-cultures of rat embryonic day 14 (E14) ventral mesencephalon (VM) and E21 striatum, we have described the developmental changes in (i) dopamine (DA) neurochemistry; (ii) numbers of DA neurons; and (iii) protein expression of tyrosine hydroxylase (TH), DA transporter (DAT), and glutamic acid decarboxylase (GAD 65/67), over 17 days in vitro (DIV). Co-cultures demonstrated changes in DA development similar to those observed in vivo. The numbers of VM DA neurons remained relatively constant, while levels of VM DA progressively increased through 10 DIV. After 3 DIV, the levels of striatal DA increased substantially, through 10 DIV. Tissue levels of DA metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) reflected changes in tissue DA concentrations, indicating that release and metabolism of DA are similar to these characteristics observed in vivo. Western blot analysis of TH protein expression revealed large increases in VM TH after only 3 DIV, followed by a decline in levels through 17 DIV; levels of striatal TH, in contrast, increased through this period. Additionally, DAT and GAD 65/67 expression increased, in both the VM and striatum, over 17 DIV. By 17 DIV, many measures of DA function had decreased from those assessed at 10 DIV, thus providing an approximate limit to the effective duration of use of this co-culture model. Our results provide a much-needed description of the neurochemical changes that occur during the maturation of VM and striatum in organotypic co-cultures. Additionally, these results provide a foundation for future studies to assess toxic challenges of the developing nigrostriatal DA system, in vitro.

Dopaminergic Bases of Polychlorinated Biphenyl-Induced Neurotoxicity

Publisher Summary This chapter reviews the work that demonstrates that the orthosubstituted congeners type of polychlorinated biphenyls (PCBs) are neurologically active and may induce neurochemical changes in both the adult and developing central nervous system (CNS) by different mechanisms than the coplanar congener type of PCBs. The PCBs are members of a large class of persistent environmental contaminants known as halogenated aromatic hydrocarbons (HAHs). Data suggesting that PCBs are neurotoxicants are based primarily on two major series of studies. The first are epidemiological and suggest an association between perinatal exposure of humans to PCBs and delays in motor reflex development and cognitive dysfunctions. The second series describes behavioral and neurochemical changes in nonhuman primates and laboratory rodents exposed either perinatally or as adults to PCBs. This chapter discusses in detail the neurochemical effects of perinatal exposure to PCBs. The neurochemical effects of PCBs in in vitro preparations are also described. The chapter also examines the neurochemical changes seen in rodents, nonhuman primates, and tissue culture preparations exposed to PCBs and describes the possible biochemical mechanisms responsible for the changes in dopaminergic function.