The majority of our research effort in the past year has been on a class of flame retardants, polybrominated diphenyl ethers (PBDE). PBDE's are produced commercially as mixtures based on bromine content. For instance, Great Lakes DE-71tm (DE-71) is a commercial mixture containing 71% bromine used primarily as a flame retardant in polyurethane foams. DE-71 contains penta-, tetra- , and hexa-brominated diphenyl ethers. PBDE's are found in mammalian tissues and fluids, including human adipose, serum, and milk. The most prevalent congeners in human samples are BDE-47 (a tetraBDE), BDE-99, and BDE-100 (both pentaBDE's). These are also the main congeners in the DE-71 commercial mixture. The major hexaBDE is BDE-153 and while it is a minor component compared to the tetra- and penta-congeners, it is highly lipophilic and may be more persistent in vivo. The NTP is currently designing a 2-year bioassay for the DE-71 mixture. PBDE's have relatively low acute toxicity and appear to be non-mutagenic. However, PBDE congeners are structurally similar to TCDD and PCB's and have been considered to have mechanisms of toxicity in common with those chemicals. Additionally, BDE-47 and its hydroxylated metabolites are structurally similar to thyroxin and may interfere with thyroid hormone controlled pathways. We have nearly completed the ADME studies of the congeners, BDE-47, -99, and -153. The studies have covered a wide range of doses from 0.1 to 1000 umol/kg. 14C-BDE-47, -99 and -153 are well absorbed (70 to 85%) from oral administration of a corn oil formulation. Tissue distribution is linear over the 10,000-fold range of doses, at least for the larger tissues. While adipose tissue was a major depot for 14C from all three congeners, BDE-47 was distributed to the thyroid to a greater extent than BDE-99 or -153. The similarity between TCDD, PCBs and PBDEs was pointed out earlier. A dispositional feature of TCDD and planar PCBs is a ratio of liver to fat concentrations of more than 1. All three PBDE congeners have liver to fat ratios less than 1. While there may appear to be a similarity among the three classes of chemicals when drawn in 2-dimensions, the 3-dimensional structures are quite different. Using molecular mechanics programs to find conformational energy minima results in structures where the two aromatic rings of biphenyl ethers are more or less perpendicular. The calculations also indicate that forcing the rings into coplanarity requires too much energy for them to bind to receptors that require planar molecules like TCDD. In spite of the evidence that PBDEs do not have similar properties, there remain the reports of TCDD-like biological activity of commercial flame-retardant products, especially induction of cytochromes CYP1A1/2. To explore this apparent contradiction, differential gene expression studies on animals treated with DE71, or one of the congeners were conducted. Using RT-PCR, upregulation of CYP 1A, CYP 2B and CYP 3A was determined. DE71 treatment did cause a slight upregulation of CYP 1A. On a molar basis DE71 is about 10,000 times less potent than the coplanar PCB-126. Of the three congeners only BDE-153 caused appreciable upregulation, but the response was less than the mixture. The congeners did cause upregulation of CYP 2B and to a lesser extent, CYP 3A. The ability to upregulate CYP 1A appears to be due to brominated dibenzofurans present in the DE71 as impurities. These dibenzofurans are present in the individual congeners also, but at much lower concentrations. The highest concentration was in BD-153, the congener that caused the most upregulation of the three. The PBDEs are not likely to cause biological or toxicological effect similar to TCDD or planar PCBs. Most analyses of human tissue have been focused on PBDE's. Evidence presented above implies that analysis of the tissues for brominated dibenzofurans and dioxins should also be considered. The metabolism and disposition of 14C-labeled juglone in male F344 rats following oral, intravenous, and dermal administration have been studied. Juglone (5-hydroxy-1,4-naphthoquinone) is present in the hulls and leaves black walnut (Juglans nigra) and related species. Black walnut extract (BWE) is marketed as an herbal medicine to treat various ailments but walnut hull products are listed as """"""""Unapproved Herbs"""""""" in the German Commission E Monographs because of potential mutagenic and carcinogenic effects of juglone. BWE has been nominated to the National Toxicology Program for study, including the metabolism and disposition of juglone. Quinones are reactive compounds due to their redox activity and electrophilicity. Quinones can undergo one- or two-electron reduction to give semiquinone anion radicals or hydroquinones, respectively. Once the hydroquinones are generated, the hydroxyl groups undergo an enzyme-catalyzed conjugation with glucuronide or sulfate. These glucuronide and sulfate conjugates remain as reduced species and therefore their formation results in detoxification. Reductive 1,4-addition of certain quinones with thiols such as glutathione (GSH) takes place spontaneously to form GSH-hydroquinones. GSH conjugates of 1,4-naphthoquinones autoxidize at faster rates than the parent hydroquinones, so the GSH conjugates of quinones still retain redox activity and electrophilicity. Quinone-thioethers from conjugation of quinones with GSH or N-acetylcysteine have been shown to induce nephrotoxicity when administered intravenously. Metabolic reduction of quinones is not well studied; only a few examples can be found in the literature. Disposition studies of 14C-labeled juglone in male F344 rats following oral, intravenous, and dermal administration (0.1 to 10 mg/kg) have been conducted to provide information on the fate of this reactive quinone when administered through three different routes. Approximately 40-50% of an oral dose (0.1 to 10 mg/kg) and less than 20% of the dermal dose (4 mg/kg) were absorbed within 24 h. Most of the oral dose was excreted in feces and urine within 24 h and only 1-3% remained in the tissues. High concentrations of juglone-derived radioactivity were found in kidney for all three dosing routes. The accumulation in kidney can be attributed to covalent binding to cytosolic protein. Five metabolites were identified in urine of rats treated with an oral dose: a 1,4,5-trihydroxynaphthalene di-glucuronide, a 1,4,5-trihydroxynaphthalene mono-glucuronide mono-sulfate, 2-sulfo-2,3-dihydrojuglone, a 4,8-dihydroxy-1-tetralone mono-glucuronide, and a 1,4,5-trihydroxynaphthalene mono-glucuronide. Liver microsomal incubations of juglone in the presence of NAD(P)H and UDP-glucuronic acid (UDPGA) gave rise to two 1,4,5-trihydroxynaphthalene monoglucuronides. No products from a GSH adduct pathway , e,g, mercapturic acids, were identified. Juglone is a reactive chemical and the toxicity observed will depend on route of exposure.
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