Environmental Toxicokinetics and Toxicology

National Institute Of Environmental Health Sciences

Investigators

Linked publications & trials

Abstract

1)The flame retardant Tetrabromobisphenol A (TBBPA) is present in the epoxy resin of circuit boards and has been found at significant levels in house dust. Studies performed by the National Toxicology Program have indicated a link between higher exposure levels and uterine tumors in rats, and TBBPA was listed as a probable carcinogen by the International Agency for Research on Cancer (IARC) in 2016. Evidence has also accumulated that TBBPA may have an endocrine disrupting function. In previous studies using Wistar Han rats chronically treated with 250 mg/kg/d for 5 days, our lab has linked TBBPA exposure to disruption in estrogen homeostasis, along with thyroid and immune system dysfunction. A potential effect on expression of the genes comprising the circadian clock was also discovered upon RNA-Seq analysis of liver and uterus from these TBBPA- and vehicle-treated rats. To investigate this effect, a circadian study was undertaken. Female Wistar Han rats were exposed to 250 mg/kg of TBBPA or vehicle for 5 consecutive days, then tissues were collected at 4-hour intervals and RNA was extracted. Expression levels of the core circadian genes Clock circadian regulator (Clock), aryl hydrocarbon nuclear translocator-like (Arntl or Bmal1), neuronal PAS domain protein 2 (Npas2), period circadian regulator 1, 2 and 3 (Per1, Per2, and Per3), and cryptochrome circadian regulator 1 and 2 (Cry1, Cry2) and circadian-related genes retinoic acid receptor-related orphan receptor a, b and c (Rora, Rorb, Rorc), nuclear receptor subfamily 1 group D member 1 (Nr1d1, Rev-erb), casein kinase 1d (Csnk1d) and 1e (Csnk1e), aryl hydrocarbon receptor (Ahr), and aryl hydrocarbon receptor nuclear translocator (Arnt) were measured in liver and uterus using droplet digital PCR (ddPCR). Protein levels in nuclear extracts are also being determined using a SimpleWestern (ProteinSimple) system for comparison to gene expression values.Gene expression results have shown differential effects in liver and uterus, including decreased amplitude, phase shift, and suppression of cyclicity, which appear to be gene- and tissue-specific. Some potential phase shift has also been detected in preliminary protein level data. These perturbations of the circadian cycle could affect expression of numerous genes which are reliant on circadian signals to regulate expression, including those controlling metabolism and the reproductive cycle.Gene expression data has been completed and presented as a poster at SOT. Protein levels are currently being determined in nuclear extracts and will be completed within one to two months, at which point the data will be correlated and prepared as a poster for SOT and a manuscript for publication and the project will be complete. 2)Studies of the flame retardant 2,4,6-tribromophenol (TBP) 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. TBP has recently been shown to accumulate in placenta by unknown mechanisms. However, 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 oral and dermal bioavailability, detoxication and activation pathways, target tissues and accumulation potential, and rates and routes of dose excretion. The disposition of TBP in pregnant and nursing rats is under investigation, with preliminary findings prepared for presentation at a scientific meeting in FY2020 and a manuscript describing these data was published in FY2020. This project is completed. 3)Studies of efflux transporter activity and function in barrier tissues following xenobiotic exposure to GenX Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoate (CAS No. 62037-80-3, GenX), 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 blood-brain barrier (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 (1, 10, 100 nM, or 1 M). 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 M. At 100nM, GenX caused a decrease in BCRP activity over time (0 to 6 hours). 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. These studies are complete and resulted in a published manuscript in EHP. 4)Work from our laboratory indicates that endogenous or exogenous nitric oxide (NO) significantly modulates activities of topoisomerases and ABC transporters in human tumor cells by nitrosylating the sulfhydryl groups of proteins. Recently, we found that GenX, a pollutant found in drinking water, inhibits the transporter activities of both Pgp and BCRP without changing the expression of the transporters. It is interesting to note that the treatment of MDR cells with nanomolar concentrations of GenX reversed Adriamycin resistance in a Pgp-expressing tumor cells. Our preliminary studies suggest that effects of GenX on P-gp transport activity may be due to formation of NO from nitric oxide synthase (NOS) as inhibition of NOS blocks the activities of GenX in isolated rat brain capillaries. Our ongoing work is focused on elucidating the roles of NOS and NO in the mechanism of actions in tumor cytotoxicity as well as in deciphering actions of GenX both in vitro and in vivo models. This work has produced 6 publications. 5)The blood-brain barrier (BBB) is a network of specialized brain vasculature that separates the central nervous system from peripheral circulation. Barrier function relies on physical and biochemical features that are represented by tight junction proteins and ABC transporters, respectively. Well-studied members of the ATPbinding cassette (ABC) transporter family, including P-glycoprotein (P-gp, ABCB1), Breast Cancer Resistant Protein (BCRP, ABCG1) and Multidrug Resistant Protein (MRP2, ABCC2), are highly expressed at the BBB. Our laboratory has shown that xenobiotics can also disrupt and modulate these pumps, thereby increasing the risk of BBB dysfunction. The present and traditional method of examining the effects of xenobiotics on important ABC transporters involves measuring the luminal accumulation of fluorescent transporterspecific substrates in freshly isolated rat brain capillaries by confocal microscopy. Steady-state luminal fluorescence is quantified in digitally acquired images to assess transport activity, but this procedure is time-intensive and subject to human bias. To measure luminal fluorescence in rat brain capillaries efficiently and objectively we have developed, an alternative method using an automated confocal based instrument coupled with machine module programing. This method is presently being optimized for future use as a mid- to high-through put screen of xenobiotics in cells and rat capillaries.