Heldur Hakk

Toxicokinetics of the Flame Retardant Hexabromocyclododecane Gamma: Effect of Dose, Timing, Route, Repeated Exposure, and Metabolism

Alpha-hexabromocyclododecane (α-HBCD) is an emerging persistent organic pollutant present in the hexabromocyclododecane (HBCD) commercial mixture. HBCD is used as an additive flame retardant in a wide variety of household consumer products. Three main stereoisomers, alpha (α), beta (β), and gamma (γ), comprise roughly 10, 10, and 80% of the mixture, respectively. Despite its small contribution to HBCD global production and usage, α-HBCD is the major stereoisomer found in wildlife and human tissues including breast milk and blood in North America, European Union, and Asia. No mammalian or human data are currently available regarding the toxicokinetics of α-HBCD. This study was conducted in an effort to fully characterize the absorption, distribution, metabolism, and elimination of α-HBCD following a single and repeated exposure with respect to dose, time, and route of administration in female C57BL/6 mice. Results indicate that ∼90% of the administered dose (3 mg/kg) was absorbed after oral exposure. Disposition was (1) dictated by lipophilicity, as adipose, liver, muscle, and skin were major depots and (2) was dose dependent with nonlinear accumulation at higher doses. Elimination, both whole-body and from individual tissues, was biphasic. α-HBCD-derived radioactivity was excreted in the feces as parent and metabolites, whereas urine only contained metabolites. Presence of polar metabolites in the blood and urine were a major factor in determining the rapid initial whole-body half-life after a single oral exposure. Initial half-lives were ∼1-3 days and much longer terminal half-lives of 17 days were observed, suggesting the potential for α-HBCD bioaccumulation. A 10-day repeated study supports α-HBCD bioaccumulation potential. Stereoisomerization previously observed after exposure to γ-HBCD was not seen after exposure of α-HBCD. The toxicokinetic behavior reported here has important implications for the extrapolation of toxicological studies of the commercial HBCD mixture to the assessment of risk of α-HBCD which is the major stereoisomer found in wildlife and people.

Novel and Distinct Metabolites Identified Following a Single Oral Dose of α- or γ-Hexabromocyclododecane in Mice

The metabolism of α- and γ-hexabromocyclododecane (HBCD) was investigated in adult C57BL/6 female mice. α- or γ-[(14)C]HBCD (3 mg/kg bw) was orally administered with subsequent urine and feces collection for 4 consecutive days; a separate group of mice was dosed and sacrificed 3 h postexposure to investigate tissue metabolite levels. Extractable and nonextractable HBCD metabolites were quantitated in liver, blood, fat, brain, bile, urine, and feces and characterized by LC/MS (ESI-). Metabolites identified were distinct between the two stereoisomers. In mice exposed to α-HBCD, four hydroxylated metabolites were detected in fecal extracts, and one of these metabolite isomers was consistently characterized in liver, brain, and adipose tissue extracts. In contrast, fecal extracts from mice exposed to γ-HBCD contained multiple isomers of monohydroxy-pentabromocyclododecene, dihydroxy-pentabromocyclododecene, and dihydroxy-pentabromocyclododecadiene, while in liver and adipose tissues extracts only a single monohydroxy-pentabromocyclododecane metabolite was observed. Both stereoisomers were transformed to metabolites which formed covalent bonds to proteins and/or lipids in the gut as suggested by high fecal nonextractables. The presence of tissue- and excreta-specific metabolic products after in vivo exposure to the two main HBCD stereoisomers supports previous toxicokinetic studies indicating that these two stereoisomers are biologically distinct. The distinct metabolic products identified in this study have the potential to aid in the identification of stereoisomer-specific HBCD exposures in future biomonitoring studies.

Absorption, distribution, metabolism and excretion (ADME) study with 2,2′,4,4′,5,6′-hexabromodiphenyl ether (BDE-154) in male Sprague–Dawley rats

A metabolism study of orally administered 2,2′,4,4′,5,6′-hexabromodiphenyl ether (BDE-154; 11.3 μmoles kg−1) was conducted in conventional and bile duct-cannulated male Sprague–Dawley rats. In conventional rats, approximately 31% of the radiolabelled dose was retained at 72 h, and lipophilic tissues were the preferred sites for disposition. Urinary excretion of BDE-154 was very low (1.0%), and parent compound was detected. Cumulative biliary excretion was 1.3%, and glutathione conjugates were suggested. Over 62% of the dose in conventional male rats was excreted in faeces, and was composed of parent compound (7.3%), free metabolites (13.1%), and covalently bound residues (41.4%). Faecal metabolites characterized by gas chromatography/mass spectrometry included multiple isomers of monohydroxylated hexa-/penta-/tetrabromodiphenyl ethers, and di-hydroxylated hexa/pentabromodiphenyl ethers. The adipose tissue 14C was extractable BDE-154, but 40% of liver 14C was bound to macromolecules. The study demonstrated the importance of performing individual polybrominated diphenyl ether (PBDE) metabolism studies to understand fully PBDE pharmacokinetics.

Tissue distribution, excretion, and metabolism of 1,2,7,8-tetrachlorodibenzo-p-dioxin in the rat.

A tissue distribution, excretion, and metabolism study was conducted using a relatively non-toxic dioxin congener, i.e., 1,2,7,8-tetrachlorodibenzo-p-dioxin (1278-TCDD), to gain a better understanding of mammalian metabolism of dioxins. Conventional, bile duct cannulated, and germ free male rats were administered mg/kg quantities as a single oral dose. Elimination of 1278-TCDD was largely complete by 72 h. Distribution of [14C]1278-TCDD was low in all tissues examined. Metabolites were identified in urine, bile, and feces by negative ion FAB-MS and 1H-NMR, or GC/MS. The major fecal metabolite was a NIH-shifted hydroxylated TCDD. The bile contained a glucuronide conjugate of this hydroxy TCDD, and a diglucuronide conjugate of a dihydroxy-triCDD. The major metabolites in urine were glucuronide and sulfate conjugates of 4,5-dichlorocatechol.

Identification of compounds in an HPLC fraction from female extracts that elicit mating responses in male screwworm flies,Cochliomyia hominivorax

When hexane extracts of mature screwworm females were chromatographed on a silica gel column, mating stimulant activity was concentrated in a fraction that eluted with hexane-ether (94∶6, v/v). Separation of this fraction with HPLC (acetonitrile-acetone; 60∶40, isocratic) resulted in a chromatogram of some 20 peaks. Only peaks 4–11 elicited mating responses. Peaks 5–10 had most of the activity, with peak 8 producing the highest response. Sixteen compounds were characterized from peak 8 by gas chromatography-mass spectrometry: six unbranched secondary acetates (C31H62O2); seven previously unreported methyl-branched secondary acetates (C32H64O2); one unbranched ketone (C31H62O); and one methyl-branched ketone (C32H64O). The isomeric acetates were not completely resolved from each other by capillary gas chromatography (CGC) on methyl silicone columns. The sixteenth compound was an aldehyde (C30H60O) that was present only in occasional peak 8 preparations. These compounds and several derivatives were characterized by capillary gas chromatography-mass spectrometry (CGC-MS). The position of the acetate group was ascertained by conversion to a keto group or by replacement of the acetate with a methyl group. Pheromone activity was not observed in peaks trapped either from CGC or by recombination of the trapped CGC peaks from HPLC peak 8. This apparent loss of activity from CGC peaks or from TLC cannot currently be explained.

ALKANES FROM SURFACE LIPIDS OF SUNFLOWER STEM WEEVIL, Cylindrocopturus adspersus (LECONTE)

The stem weevil,Cylindrocopturus adspersus (LeConte) (Coleoptera: Curculionidae) yields 3% of its body weight as extractable lipids (40 μg/ weevil). The alkane fraction was composed ofn-alkanes (38%) and branched alkanes (62%). The compounds were characterized by gas chromatography-mass spectrometry (GC-MS). The chromatogram contained several single-component peaks (9 of 25). Only seven dimethylalkanes were isolated (17.8%): 9,19- and 9,21-dimethylheptacosane; 9,19- and 9,21-dimethylnonacosane; 9,21- and 11,21-dimethylhentriacontane; and 11,21-dimethyltritriacontane. Important methylalkanes were: 2-methyltetra- and hexacosanes and 10-methylhexa- and octacosanes. Late-eluting gas chromatography peaks were composed of simple alkane mixtures or a single component.

Multicomponent attractant for female screwworm flies, Cochliomyia hominivorax, in bovine blood

An olfactometer bioassay was used to follow attractant for screwworm flies,Cochliomyia hominivorax, in steam distillates of bovine blood under different distillation and storage conditions and after HPLC separation of components in a water-methanol gradient. In addition, fly responsiveness was examined in relation to sex and ovarian stage. Gravid and vitellogenic nullipars were attracted to the blood, although the former predominated four to one. Males did not respond at a dose that attracted 76% of gravid females. Maximum attractiveness occurred when distillate was stored in sealed glass ampoules. An argon atmosphere made storage at ambient temperatures feasible, but offered no advantage during storage at ca. −60°C or during distillation. The HPLC separation produced four fractions that duplicated the attractiveness of the distillate when recombined but showed little activity when presented as two-fraction, and most three-fraction, mixtures. Availability of the HPLC fractions for combination with other samples will facilitate location via bioassay of attractant components in samples obtained from subsequent or alternate isolations that preserve only one or two elements of the multicomponent mixture.

Metabolism of [14C]1,2,7,8-tetrachlorodibenzo-p-dioxin in a ruminating Holstein calf

1. [UL-7,8-ring 14C]-1,2,7,8-tetrachlorodibenzo-p-dioxin (1278-TCDD) was administered orally to a ruminating Holstein bull calf (43.6 kg; 1.2 mg kg-1 body weight). Urine and faeces were collected daily for 96 h, while blood was sampled at multiple time points. Tissues were removed for combustion analysis. 2. Each tissue contained < 0.6% of the dose at 96h. Tissues with highest levels of 1278-TCDD, as a percentage of administered dose, were the large and small intestine, rumen, liver and carcass. 3. Urinary excretion accounted for 10.6% of the dose, and faecal excretion accounted for 81.6% of the administered dose. The major urinary and faecal metabolites were isolated and characterized by mass spectrometry and 1H-NMR. 4. Plasma levels of 14C peaked at 24 h, and decreased to near background at 96 h. Detectable plasma levels of 1278-TCDD were observed by 2 h. 5. A hydroxylated metabolite of 1278-TCDD was detected in calf plasma, which has the potential to interfere with thyroid hormone homeostasis.

Polybrominated Diphenyl Ethers (PBDEs) in U.S. Meat and Poultry: 2012-2013 Levels, Trends, and Estimated Consumer Exposures

ABSTRACT Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants whose use has contaminated foods and caused subsequent human exposures. To address the issue of possible human exposure, samples from a 2012–13 US meat and poultry (beef, pork, chicken, turkey) study were analysed for seven PBDEs. The mean summed concentrations of the seven BDE congeners (ΣPBDE) from beef, pork, chicken and turkey were 0.40, 0.36, 0.19, and 0.76 ng g–1 lipid weight (lw). The range of ΣPBDEs for all meat classes was 0.01–15.78 ng g–1 lw. A comparison of this study with a 2007–08 study revealed a decline in the median ΣPBDEs for all four meat classes, a reduction of 25.9% to 70.0%, with pork, chicken and turkey PBDE residues being statistically lower relative to the 2007–08 study. BDEs 47 and 99 contributed the most to the ΣPBDE concentrations, indicating likely animal exposures to the penta-BDE formulation. Based on the reported data an estimate of US consumer daily intake of PBDEs from meat and poultry was 6.42 ng day–1.