1) Anniston community health survey (ACHS-II): Follow-up study and dioxin analyses. The recruitment and sampling phases of this study are concluded. Blood was collected from 359 of 777 subjects from the original study. Laboratory analysis of these samples is underway. Preliminary results from some participants indicate a decrease over time in serum levels of many PCB congeners, especially those of lowest chlorination. New research is underway to assess the degree of DNA methylation in these subjects. 2) Toxic organic chemicals and heavy metals in blood and urine of electronic waste recycling workers and the general population in rural Vietnam. A manuscript describing the Biomonitoring of Organics and Metals in Vietnamese Female Electronic Waste Recyclers was published in FY2018. 3) Bisphenol A (BPA) pharmacokinetics: Controlled exposure study. Dermal application studies are in progress. These data will be used for further understanding of the fate of this potential endocrine disruptor in humans. Studies of the fate and toxicity of several brominated flame retardants (BFRs) and other halogenated chemicals of interest were conducted in FY2018 as follows: 1) Studies of the major flame retardant tetrabromobisphenol A (TBBPA) Wistar Han rats chronically exposed to TBBPA by the oral route have a higher incidence of uterine tumors than in controls. Studies were designed to characterize this mechanism of toxicity. A study of the fate of TBBPA in female rats did not detect evidence for accumulation of TBBPA or generation of TBBPA-derived reactive metabolites in uterine tissues. Therefore, the most likely hypothesis for the carcinogenic response involved disruption of estrogen and/or thyroid hormone homeostasis in the TBBPA-exposed rats. This hypothesis was subsequently supported by the results of molecular studies conducted in the laboratory. A manuscript describing these results was published in FY2018. Humans may also be at risk for developmental exposure to low-doses of TBBPA in the environment. Data are needed in a rodent model for extrapolation purposes; therefore, a collaborative study of prenatal and perinatal exposure to TBBPA in rats is being conducted with other NIEHS researchers. 2) Studies of the major flame retardant 2,4,6-tribromophenol TBP has been long used as a flame retardant and wood fungicide and is both a precursor for, and degradation product of, PBDEs. TBP exposure in human populations is ubiquitous. Adequate toxicological evaluations for TBP is lacking and toxicokinetic studies are being conducted in this laboratory to provide supporting data for designing and interpreting toxicity studies and risk assessments for TBP. These studies presently include investigations of the fate of TBP in rodents by different exposure routes, with emphasis on determining bioavailability, detoxication and activation pathways, target tissues and accumulation potential, and rates and routes of dose excretion. 3) Studies of efflux transporter activity and function in barrier tissues following xenobiotic exposure to TBBPA The blood-brain barrier and the placental barrier protect the brain and the developing fetus. Earlier studies indicate that signal transduction pathways regulate the activity and expression of numerous transporters that line the lumen of the microvessels in the brain and the placenta. Our overall research objective is to determine if xenobiotics cross these barriers and/or modulate the ABC transporters. Current studies are focused on the effects of the brominated flame retardants TBBPA on transporter function and expression. Ex vivo experiments in rats have been performed to measure both expression and transport activity following exposure to TBBPA. Known agonists and antagonists have been used to tentatively identify the signaling pathways involved in the regulation of two canonical transporters, P-glycoprotein (Pgp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) following exposure in culture. We found that TBBPA significantly increased P-glycoprotein transport activity 50%, at 1.0-100nM in 1-4 hours of exposure. We also measured BCRP and Mrp2 transport activity following TBBPA exposure. BCRP transport decreased while Mrp2 transport was unchanged. Inhibition of either transcription or translation blocked the TBBPA increases in P-glycoprotein transport. Western blots indicate that capillaries exposed to TBBPA contain higher levels of P-glycoprotein protein. Pharmacological inhibition of the nuclear receptor, PPAR-g, completely blocked the TBBPA dependent increases in P-glycoprotein transport and expression. In vivo studies have been used to validate ex vivo findings, indicating this is a robust and biologically relevant phenomenon. Additional studies are being designed to designed to measure the entry of radiolabeled substrates in brain and placenta. 4) Studies of efflux transporter activity and function in barrier tissues following xenobiotic exposure to TBP 2,4,6-Tribromophenol (TBP) is a brominated chemical used in the production of flame retardant epoxy resins, and as a wood preservative. TBP is a bio-active and bio-concentrated endocrine disruptor that interferes with estrogen and thyroid hormone signaling. In this study, we examined the effect of TBP exposure on the transport activity of three well-characterized ABC efflux transporters: P-gp, BCRP, and MRP2 in freshly isolated rat brain microvessels. We found TBP exposure resulted in a time and dose dependent decrease in P-gp and BCRP transport activity. We saw no change in MRP2 transport activity under identical conditions, indicating selectivity and capillary integrity. Known agonists and antagonists have been used to tentatively identify the signaling pathways involved in the regulation of P-glycoprotein and BCRP following exposure in culture. Additional studies are being designed to validate ex vivo findings using an in vivo model designed to measure the entry of radiolabeled substrates in brain and placenta. Decreases in transporter activity at the blood-brain barrier can reduce neuroprotection and increase CNS exposure to potentially toxic endobiotics and xenobiotics. 5) Studies of efflux transporter activity and function in barrier tissues following xenobiotic exposure to GenX Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate, is a chemical precursor in the production of polytetrafluoroethylene and is a replacement for perfluorooctanoic acid. Based on a high-dose (300 mg/kg) toxicokinetic study, GenX is unlikely to be metabolized, has high oral bioavailability, and is almost exclusively excreted via urine. Based on its high bioavailability, persistence, likely human exposure, and unknown CNS toxicity, we investigated the potential for GenX to modulate the BBB. We investigated the effects of GenX on three well-characterized efflux transporters (P-gp, BCRP, and MRP2) by exposing rat brain capillaries ex vivo to increasing concentrations of GenX. In a steady-state transport model, we found that GenX decreased the transport activity of P-gp in a linear dose- and time- dependent manner, and that the effect was irreversible. BCRP activity decreased nonmonotonically from 1 nM to 100 nM concentrations of GenX, but it had no effect at the highest dose of 1 uM. At 100nM, GenX caused a periodically oscillating decrease in BCRP activity over time. GenX had no effect on activity of MRP2 at the concentrations tested, indicating that the effects seen on P-gp and BCRP were specific and that the capillaries were not compromised structurally by the chemical. Decreased P-gp and BCRP activity may reduce the neuroprotective function of the BBB and alter pharmacokinetics of their substrates, potentially exposing the CNS to toxic endogenous and exogenous substrates.
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