The novel pyridazine pyrazolecarboxamide insecticide dimpropyridaz inhibits chordotonal organ function upstream of TRPV channels.

BACKGROUND: Pyridazine pyrazolecarboxamides (PPCs) are a novel insecticide class discovered and optimized at BASF. Dimpropyridaz is the first PPC to be submitted for registration and controls many aphid species as well as whiteflies and other piercing-sucking insects. RESULTS: Dimpropyridaz and other tertiary amide PPCs are proinsecticides that are converted in vivo into secondary amide active forms by N-dealkylation. Active secondary amide metabolites of PPCs potently inhibit the function of insect chordotonal neurons. Unlike Group 9 and 29 insecticides, which hyperactivate chordotonal neurons and increase Ca2+ levels, active metabolites of PPCs silence chordotonal neurons and decrease intracellular Ca2+ levels. Whereas the effects of Group 9 and 29 insecticides require TRPV (Transient Receptor Potential Vanilloid) channels, PPCs act in a TRPV-independent fashion, without compromising cellular responses to Group 9 and 29 insecticides, placing the molecular PPC target upstream of TRPVs. CONCLUSIONS: PPCs are a new class of chordotonal organ modulator insecticide for control of piercing-sucking pests. Dimpropyridaz is a PPC proinsecticide that is activated in target insects to secondary amide forms that inhibit the firing of chordotonal organs. The inhibition occurs at a site upstream of TRPVs and is TRPV-independent, providing a novel mode of action for resistance management. © 2023 BASF Corporation. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

2,4,6-Tribromophenol Disposition and Kinetics in Pregnant and Nursing Sprague Dawley Rats.

2,4,6-Tribromophenol (TBP, CAS no. 118-79-6) is a brominated chemical used as a precursor, flame retardant, and wood antifungal agent. TBP is detected in environmental matrices and biota, including human breast milk, placenta, and serum. To address reports of TBP accumulation in human placenta and breast milk, studies were conducted to characterize TBP disposition and toxicokinetics in timed-pregnant or nursing Sprague Dawley rats following a single oral dose to the dam. Animals were administered [14C]-TBP (10 µmol/kg, 25 µCi/kg, 4 ml/kg) by gavage on gestation day 12 and 20, or postnatal day 12 and serially euthanized between 15 min and 24 h for collection of blood and tissues from the dam and fetuses/pups. Observed plasma TBP Cmax (3 and 7 nmol/ml) occurred at 15 min in both GD12 and GD20 dams while Cmax (3 nmol/ml) was observed at 30 min for PND12 dams. Concentrations in tissues followed plasma concentrations, with kidneys containing the highest concentrations at 30 min. GD12 litters contained a sustained 0.2%-0.3% of the dose (5-9 nmol/litter) between 15 min and 6 h while GD20 fetuses (2%-3%) and placentas (0.3%-0.5%) had sustained levels between 30 min and 12 h. The stomach contents (approx. 1 nmol-eq/g, 6-12 h), livers (0.04-0.1 nmol-eq/g) and kidneys (0.1-0.2 nmol-eq/g) of PND12 pups increased over time, indicating sustained exposure via milk. Systemic exposure to TBP and its metabolites occurs in both the directly exposed mother and the indirectly exposed offspring and is rapid and persistent after a single dose in pregnant and nursing rats.

Effect of GenX on P-Glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance-Associated Protein 2 at the Blood-Brain Barrier.

BACKGROUND: Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX) is a replacement for perfluorooctanoic acid in the production of fluoropolymers used in a variety of consumer products. GenX alters fetal development and antibody production and elicits toxic responses in the livers and kidneys of rodents. The GenX effect on the blood-brain barrier (BBB) is unknown. The BBB protects the brain from xenobiotic neurotoxicants and harmful endogenous metabolites. OBJECTIVES: We aimed to investigate the effects of GenX on the transport activity and expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) at the BBB. METHODS: Transporter activities were measured in isolated rat brain capillaries by a confocal microscopy-based method. ATPase (enzymatic hydrolysis of adenosine triphosphate to inorganic phosphate) levels were measured in vitro. Western blotting determined P-gp and BCRP protein levels. Cell survival after GenX exposure was determined for two human cell lines. RESULTS: Nanomolar levels of GenX inhibited P-gp and BCRP but not MRP2 transport activities in male and female rat brain capillaries. P-gp transport activity returned to control levels after GenX removal. GenX did not reduce P-gp- or BCRP-associated ATPase activity in an in vitro transport assay system. Reductions of P-gp but not BCRP transport activity were blocked by a peroxisome proliferator-activated receptor γ (PPARγ) antagonist. GenX reduced P-gp and BCRP transport activity in human cells. CONCLUSION: In rats, GenX at 0.1-100 nM rapidly (in 1-2 h) inhibited P-gp and BCRP transport activities at the BBB through different mechanisms. PPARγ was required for the GenX effects on P-gp but not BCRP transport activity. https://doi.org/10.1289/EHP5884.

2,4,6-Tribromophenol Exposure Decreases P-Glycoprotein Transport at the Blood-Brain Barrier.

2,4,6-Tribromophenol (TBP, CAS No. 118-79-6) is a brominated chemical used in the production of flame-retardant epoxy resins and as a wood preservative. In marine environments, TBP is incorporated into shellfish and consumed by predatory fish. Food processing and water treatment facilities produce TBP as a byproduct. 2,4,6-Tribromophenol has been detected in human blood and breast milk. Biologically, TBP interferes with estrogen and thyroid hormone signaling, which regulate important transporters of the blood-brain barrier (BBB). The BBB is a selectively permeable barrier characterized by brain microvessels which are composed of endothelial cells mortared by tight-junction proteins. ATP-binding cassette (ABC) efflux transporters on the luminal membrane facilitate the removal of unwanted endobiotics and xenobiotics from the brain. In this study, we examined the in vivo and ex vivo effects of TBP on two important transporters of the BBB: P-glycoprotein (P-gp, ABCB1) and Multidrug Resistance-associated Protein 2 (MRP2, ABCC2), using male and female rats and mice. 2,4,6-Tribromophenol exposure ex vivo resulted in a time- (1-3 h) and dose- (1-100 nM) dependent decrease in P-gp transport activity. MRP2 transport activity was unchanged under identical conditions. Immunofluorescence and western blotting measured decreases in P-gp expression after TBP treatment. ATPase assays indicate that TBP is not a substrate and does not directly interact with P-gp. In vivo dosing with TBP (0.4 µmol/kg) produced decreases in P-gp transport. Co-treatment with selective protein kinase C (PKC) inhibitors prevented the TBP-mediated decreases in P-gp transport activity.

Tetrabromobisphenol A (TBBPA) Alters ABC Transport at the Blood-Brain Barrier.

Tetrabromobisphenol A (TBBPA, CAS No. 79-94-7) is a brominated flame retardant used in 90% of epoxy coated circuit boards. Exposures to TBBPA can induce neurotoxicity and disrupt MAPK, estrogen, thyroid, and PPAR-associated signaling pathways. Because these pathways also regulate transporters of the central nervous system barriers, we sought to determine the effect of TBBPA on the expression and activity of 3 ATP binding cassette (ABC) transporters of the blood-brain barrier (BBB). Using a confocal based assay, we measured the ex vivo and in vivo effects of TBBPA on P-glycoprotein (P-gp), breast cancer resistant protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) transport activity in rat brain capillaries. Our rationale for using a rat model was based on tissue availability, ease of handling, and availability of historical TBBPA toxicokinetic data. We found that TBBPA (1-1000 nM) exposure had no significant effect on multidrug resistance-associated protein 2 transport activity in either sex, suggesting TBBPA does not compromise the physical integrity of the BBB. However, low concentrations of TBBPA (1-100 nM) significantly decreased breast cancer resistant protein transport activity in both sexes. Additionally, TBBPA exposures (1-100 nM), elicited a sex-dependent response in P-gp transport: increasing transport activity in males and decreasing transport activity in females. All TBBPA dependent changes in transport activity were dose- and time-dependent. Inhibitors of either transcription or translation abolished the TBBPA dependent increases in male P-gp transport activity. Western blot and immunofluorescent assays confirmed the TBBPA dependent P-gp increases expression in males and decreases in females. Antagonizing PPAR-γ abolished the TBBPA dependent increases in males but not the decreases in females. However, the decreases in female P-gp transport were blocked by an ER-α antagonist. This work indicates that environmentally relevant concentrations of TBBPA (1-100 nM) alter ABC transporter function at the BBB. Moreover, permeability changes in the BBB can alter brain homeostasis, hinder central nervous system drug delivery, and increase the brain's exposure to harmful xenobiotic toxicants.

2,4,6-Tribromophenol Disposition and Kinetics in Rodents: Effects of Dose, Route, Sex, and Species.

2,4,6-tribromophenol (TBP, CAS No. 118-79-6) is widely used as a brominated flame retardant and wood antifungal agent. TBP is frequently detected in environmental matrices, biota, and humans. In female SD rats, systemically available TBP (10 µmol/kg, IV) was rapidly excreted primarily via urine, with approximately 61% of the dose recovered after 4 h, and 89%-94% in 24 h; 5% was recovered in feces; and 1%-2% in blood/tissues. TBP administered to female SD rats (0.1-1000 µmol/kg) by gavage was well absorbed, with approximately 25% eliminated via urine after 4 h and approximately 88% after 24 h. Approximately 11% of a single oral dose was recovered in bile. Male SD rats and B6C3F1/J mice of both sexes had similar disposition profiles when administered a single oral dose of TBP (10 µmol/kg). Following administration, fecal recoveries varied only slightly by dose, sex, or species. TBP readily passed unchanged through both human (ex vivo only) and rat skin with between 55% and 85% of a 100 nmol/cm2 passing into or through skin. Concentrations of TBP in blood fit a two-compartment model after IV-dosing and a one-compartment model after oral dosing. Urine contained a mixture of TBP, TBP-glucuronide, and TBP-sulfate. Fecal extracts contained only parent TBP whereas bile contained only TBP-glucuronide. TBP did not appear to bioaccumulate or alter its own metabolism after repeated administration. TBP was readily absorbed at all doses and routes tested with an oral bioavailability of 23%-27%; 49% of TBP is expected to be dermally bioavailable in humans. From these data, we conclude that humans are likely to have significant systemic exposure when TBP is ingested or dermal exposure occurs.

Dermal disposition of Tetrabromobisphenol A Bis(2,3-dibromopropyl) ether (TBBPA-BDBPE) using rat and human skin.

Tetrabromobisphenol A Bis(2,3-dibromopropyl) ether (TBBPA-BDBPE) is a high production volume brominated flame retardant (BFR) used in consumer products, resulting in ubiquitous human exposure. Although the major route of exposure for this chemical is believed to be via ingestion, dermal contact is likely via contaminated dust. Independent trials of a single dose of 100 nmol/cm2 (∼1 µCi [14C]/cm2) of [14C]-radiolabeled TBBPA-BDBPE was applied to whole rat skin (in vivo) or split-thickness human and rat skin (ex vivo) to estimate in vivo human percutaneous uptake. [14C]-radioactivity was quantified to determine dermal absorption (dose retained in dosed skin) and penetrance (dose recovered in receptor fluid [ex vivo] or tissues/excreta [in vivo]) over 24 h. In vivo absorption and penetration for rat skin was 26% and 1%, with a maximum flux of 44 ± 9 pmol/cm2/h. In ex vivo rat skin, absorption and penetration and absorption values were 23% and 0.3% (flux = 26 ± 8 pmol/cm2/h). In ex vivo human skin, 53% was absorbed and penetration was 0.2% with a maximal flux of 16 ± 12 pmol/cm2/h. Computed maximal flux for in vivo human skin was 21 ± 9 pmol/cm2/h with expected total absorption of ∼80% and a penetration of <1%. HPLC-radiometric analyses of samples showed that TBBPA-BDBPE was not metabolized in ex vivo or in vivo studies. These studies indicate that TBBPA-BDBPE is likely to be dermally bioavailable even after washing and dermal contact with this chemical should be considered an important route of exposure.

Erratum: "Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity".

[This corrects the article DOI: http://dx.doi.org/10.1289/ehp.1408202.].

TBBPA disposition and kinetics in pregnant and nursing Wistar Han IGS rats.

Tetrabromobisphenol-A (TBBPA) is a brominated flame retardant (BFR) commonly used in electronics to meet fire safety standards and has the largest worldwide production of any BFR. TBBPA has been detected in human breast milk and maternal/cord serum, indicating exposure to mothers, fetuses, and breastfeeding newborns although exposure to fetuses and newborns is poorly understood. Pregnant or nursing Wistar Han IGS rats were administered [14C]-TBBPA in a single dose (25 mg/kg, 2.5 µCi/kg) and euthanized between 0.5&24 h post dose to determine disposition in pregnant and nursing rats and their pups. Systemic exposure was largely unchanged between 1&8 h post dose in pregnant rats; [14C]-radioactivity in blood varied only slightly between 0.5&8 h (2.6 ± 0.6 â†’ 2.6 ± 0.8 nmol-eq/mL) but was below the limit of detection at 24 h with an absorption half-life of 16min and elimination half-life of 17 h. Cmax was observed at 30min in lactating rats and concentrations fell steadily through 8 h. Plasma from pregnant rats contained a mixture of TBBPA and TBBPA-conjugates at 30min but only metabolites in subsequent samples. TBBPA was not detected in lactating dam plasma in this study. Placental concentrations increased through 8 h while whole-fetus Cmax occurred at 2 h post dose. In lactating animals, liver, uterus, and mammary time-concentration curves lagged slightly behind blood-concentration curves. It was clear from these studies that TBBPA is available to both the developing fetus and nursing pup following maternal exposure, and nursing pups are continuously exposed via contaminated milk produced by their mother. This research was supported in part by the Intramural Research Program of NIH/NCI.

Gene expression changes in immune response pathways following oral administration of tetrabromobisphenol A (TBBPA) in female Wistar Han rats.

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant used globally at high volumes, primarily in the epoxy resin of circuit boards. It has been detected in the environment and in humans. The National Toxicology Program found that chronic oral TBBPA treatment of 250mg/kg and higher caused an increased incidence of uterine lesions in female Wistar Han rats. The present laboratory has previously reported changes in gene expression associated with estrogen homeostasis in liver and uterine tissue of adult female Wistar Han rats after five days of gavage with 250mg/kg of TBBPA. Microarray analysis of tissue from these same TBBPA-treated rats was performed to detect additional pathways perturbed by TBBPA. Microarray analysis of uterine tissue detected downregulation of genes in pathways of the immune response following TBBPA treatment. These results, along with validation of associated gene expression changes using droplet digital PCR, are reported here. Our findings suggest mechanisms that may be related to estrogen-mediated immunosuppression.

The biological fate of decabromodiphenyl ethane following oral, dermal or intravenous administration.

1. It was important to investigate the disposition of decabromodiphenyl ethane (DBDPE) based on concerns over its structural similarities to decabromodiphenyl ether (decaBDE), high potential for environmental persistence and bioaccumulation, and high production volume. 2. In the present study, female Sprague Dawley rats were administered a single dose of [14C]-DBDPE by oral, topical or IV routes. Another set of rats were administered 10 daily oral doses of [14C]-DBDPE. Male B6C3F1/Tac mice were administered a single oral dose. 3. DBDPE was poorly absorbed following oral dosing, with 95% of administered [14C]-radioactivity recovered in the feces unchanged, 1% recovered in the urine and less than 3% in the tissues at 72 h. DBDPE excretion was similar in male mice and female rats. Accumulation of [14C]-DBDPE was observed in liver and the adrenal gland after 10 daily oral doses to rats. 4. Rat and human skin were used to assess potential dermal uptake of DBDPE. The dermis was a depot for dermally applied DBDPE; conservative estimates predict ∼14 ± 8% of DBDPE may be absorbed into human skin in vivo; ∼7 ± 4% of the parent chemical is expected to reach systemic circulation following continuous exposure (24 h). 5. Following intravenous administration, ∼70% of the dose remained in tissues after 72 h, with the highest concentrations found in lung (1223 ± 723 pmol-eq/g), spleen (1096 ± 369 pmol-eq/g) and liver (366 ± 98 pmol-eq/g); 5 ± 1% of the dose was recovered in urine and 26 ± 4% in the feces.

Disposition of the emerging brominated flame retardant, bis(2-ethylhexyl) tetrabromophthalate, in female Sprague Dawley rats: effects of dose, route and repeated administration.

1. Bis(2-ethylhexyl)-tetrabromophthalate (BEH-TEBP; CAS No. 26040-51-7; PubChem CID: 117291; MW 706.15 g/mol, elsewhere: TeBrDEPH, TBPH, or BEHTBP) is used as an additive brominated flame retardant in consumer products. 2. Female Sprague Dawley rats eliminated 92-98% of [14C]-BEH-TEBP unchanged in feces after oral administration (0.1 or 10 µmol/kg). A minor amount of each dose (0.8-1%) was found in urine after 72 h. Disposition of orally administered BEH-TEBP in male B6C3F1/Tac mice was similar to female rats. 3. Bioaccumulation of [14C]-radioactivity was observed in liver and adrenals following 10 daily oral administrations (0.1 µmol/kg/day). These tissues contained 5- and 10-fold higher concentrations of [14C]-radioactivity, respectively, versus a single dose. 4. IV-administered [14C]-BEH-TEBP (0.1 µmol/kg) was slowly eliminated in feces, with >15% retained in tissues after 72 h. Bile and fecal extracts from these rats contained the metabolite mono-ethylhexyl tetrabromophthalate (TBMEHP). 5. BEH-TEBP was poorly absorbed, minimally metabolized and eliminated mostly by the fecal route after oral administration. Repeated exposure to BEH-TEBP led to accumulation in some tissues. The toxicological significance of this effect remains to be determined. This work was supported by the Intramural Research Program of the National Cancer Institute at the National Institutes of Health (Project ZIA BC 011476).

Estimation of human percutaneous bioavailability for two novel brominated flame retardants, 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (EH-TBB) and bis(2-ethylhexyl) tetrabromophthalate (BEH-TEBP).

2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) and bis(2-ethylhexyl)tetrabromophthalate (BEH-TEBP) are novel brominated flame retardants used in consumer products. A parallelogram approach was used to predict human dermal absorption and flux for EH-TBB and BEH-TEBP. [14C]-EH-TBB or [14C]-BEH-TEBP was applied to human or rat skin at 100nmol/cm2 using a flow-through system. Intact rats received analogous dermal doses. Treated skin was washed and tape-stripped to remove "unabsorbed" [14C]-radioactivity after continuous exposure (24h). "Absorbed" was quantified using dermally retained [14C]-radioactivity; "penetrated" was calculated based on [14C]-radioactivity in media (in vitro) or excreta+tissues (in vivo). Human skin absorbed EH-TBB (24±1%) while 0.2±0.1% penetrated skin. Rat skin absorbed more (51±10%) and was more permeable (2±0.5%) to EH-TBB in vitro; maximal EH-TBB flux was 11±7 and 102±24pmol-eq/cm2/h for human and rat skin, respectively. In vivo, 27±5% was absorbed and 13% reached systemic circulation after 24h (maximum flux was 464±65pmol-eq/cm2/h). BEH-TEBP in vitro penetrance was minimal (<0.01%) for rat or human skin. BEH-TEBP absorption was 12±11% for human skin and 41±3% for rat skin. In vivo, total absorption was 27±9%; 1.2% reached systemic circulation. In vitro maximal BEH-TEBP flux was 0.3±0.2 and 1±0.3pmol-eq/cm2/h for human and rat skin; in vivo maximum flux for rat skin was 16±7pmol-eq/cm2/h. EH-TBB was metabolized in rat and human skin to tetrabromobenzoic acid. BEH-TEBP-derived [14C]-radioactivity in the perfusion media could not be characterized. <1% of the dose of EH-TBB and BEH-TEHP is estimated to reach the systemic circulation following human dermal exposure under the conditions tested. CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE: 2-Ethylhexyl 2,3,4,5-tetrabromobenzoate (PubChem CID: 71316600; CAS No. 183658-27-7 FW: 549.92g/mol logPest: 7.73-8.75 (12)) Abdallah et al., 2015a. Other published abbreviations for 2-ethylhexyl-2,3,4,5-tetrabromobenzoate are TBB EHTeBB or EHTBB Abdallah and Harrad, 2011. bis(2-ethylhexyl) tetrabromophthalate (PubChem CID: 117291; CAS No. 26040-51-7 FW: 706.14g/mol logPest: 9.48-11.95 (12)). Other published abbreviations for bis(2-ethylhexyl)tetrabromophthalate are TeBrDEPH TBPH or BEHTBP.

Disposition of the Emerging Brominated Flame Retardant, 2-Ethylhexyl 2,3,4,5-Tetrabromobenzoate, in Female SD Rats and Male B6C3F1 Mice: Effects of Dose, Route, and Repeated Administration.

2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB; MW 549.92 g/mol; CAS 183658-27-7) is a brominated component of flame retardant mixtures used as substitutes for some PBDEs. EH-TBB is added to various consumer products, including polyurethane foams, and has been detected in humans. The present study characterized the fate of EH-TBB in rodents. [14C]-labeled EH-TBB was absorbed, metabolized, and eliminated via the urine and feces following single administrations of 0.1-100 µmol/kg (∼0.05-55 mg/kg) or repeated administration (0.1 µmol/kg/day × 5-10 days) by gavage to female Hsd:Sprague DawleySD (SD) rats. Cumulative excretion via feces increased (39-60%) with dose (0.1-10 µmol/kg) with corresponding decreases in urinary excretion (54 to 37%) after 72 h. Delayed excretion of [14C]-radioactivity in urine and feces of a 100 µmol/kg oral dose was noted. Recovery was complete for all doses by 72 h. IV-injected rats excreted more of the 0.1 µmol/kg dose in urine and less in feces than did gavaged rats, indicating partial biliary elimination of systemically available compound. No tissue bioaccumulation was found for rats given 5 oral daily doses of EH-TBB. Parent molecule was not detected in urine whereas 2 metabolites, tetrabromobenzoic acid (TBBA), a TBBA-sulfate conjugate, and a TBBA-glycine conjugate were identified. EH-TBB and TBBA were identified in extracts from feces. Data from gavaged male B6C3F1/Tac mice indicated minimal sex- or species differences are likely for the disposition of EH-TBB. Approximately 85% of a 0.1 µmol/kg dose was absorbed from the gut. Overall absorption of EH-TBB is expected to be even greater at lower levels.

Disruption of estrogen homeostasis as a mechanism for uterine toxicity in Wistar Han rats treated with tetrabromobisphenol A.

Chronic oral treatment of tetrabromobisphenol A (TBBPA) to female Wistar Han rats resulted in increased incidence of cell proliferation at 250mg/kg and tumor formation in the uterus at higher doses. The present study was designed to test the hypothesis that disruption of estrogen homeostasis was a major mode-of-action for the observed effects. Biological changes were assessed in serum, liver, and the proximal (nearest the cervix) and distal (nearest the ovaries) sections of the uterine horn of Wistar Han rats 24h following administration of the last of five daily oral doses of 250mg/kg. Expression of genes associated with receptors, biosynthesis, and metabolism of estrogen was altered in the liver and uterus. TBBPA treatment also resulted in changes in expression of genes associated with cell division and growth. Changes were also observed in the concentration of thyroxine in serum and in expression of genes in the liver and uterus associated with thyroid hormone receptors. Differential expression of some genes was tissue-dependent or specific to tissue location in the uterus. The biological responses observed in the present study support the hypothesis that perturbation of estrogen homeostasis is a major mode-of-action for TBBPA-mediated cell proliferation and tumorigenesis previously observed in the uterus of TBBPA-treated Wistar Han rats.

Estimation of tetrabromobisphenol A (TBBPA) percutaneous uptake in humans using the parallelogram method.

Tetrabromobisphenol A (TBBPA) is currently the world's highest production volume brominated flame retardant. Humans are frequently exposed to TBBPA by the dermal route. In the present study, a parallelogram approach was used to make predictions of internal dose in exposed humans. Human and rat skin samples received 100 nmol of TBBPA/cm(2) skin and absorption and penetrance were determined using a flow-through in vitro system. TBBPA-derived [(14)C]-radioactivity was determined at 6h intervals in the media and at 24h post-dosing in the skin. The human skin and media contained an average of 3.4% and 0.2% of the total dose at the terminal time point, respectively, while the rat skin and media contained 9.3% and 3.5%, respectively. In the intact rat, 14% of a dermally-administered dose of ~100 nmol/cm(2) remained in the skin at the dosing site, with an additional 8% reaching systemic circulation by 24h post-dosing. Relative absorption and penetrance were less (10% total) at 24h following dermal administration of a ten-fold higher dose (~1000 nmol/cm(2)) to rats. However, by 72 h, 70% of this dose was either absorbed into the dosing-site skin or had reached systemic circulation. It is clear from these results that TBBPA can be absorbed by the skin and dermal contact with TBBPA may represent a small but important route of exposure. Together, these in vitro data in human and rat skin and in vivo data from rats may be used to predict TBBPA absorption in humans following dermal exposure. Based on this parallelogram calculation, up to 6% of dermally applied TBBPA may be bioavailable to humans exposed to TBBPA.

Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity.

BACKGROUND: Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers. OBJECTIVE: We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity. METHODS: We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis. RESULTS: We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity. CONCLUSIONS: The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity.

TITLE Disposition and kinetics of Tetrabromobisphenol A in female Wistar Han rats.

Tetrabromobisphenol A (TBBPA) is the brominated flame retardant with the largest production volume worldwide. NTP 2-year bioassays found TBBPA dose-dependent increases in uterine tumors in female Wistar Han rats; evidence of reproductive tissues carcinogenicity was equivocal in male rats. To explain this apparent sex-dependence, the disposition and toxicokinetic profile of TBBPA were investigated using female Wistar Han rats, as no data were available for female rats. In these studies, the primary route of elimination following [14C]-TBBPA administration (25, 250 or 1,000 mg/kg) was in feces; recoveries in 72 h were 95.7±3.5%, 94.3±3.6% and 98.8±2.2%, respectively (urine: 0.2-2%; tissues: <0.1). TBBPA was conjugated to mono-glucuronide and -sulfate metabolites and eliminated in the bile. Plasma toxicokinetic parameters for a 250 mg/kg dose were estimated based on free TBBPA, as determined by UV/radiometric-HPLC analyses. Oral dosing by gavage (250 mg/kg) resulted in a rapid absorption of compound into the systemic circulation with an observed Cmax at 1.5 h post-dose followed by a prolonged terminal phase. TBBPA concentrations in plasma decreased rapidly after an IV dose (25 mg/kg) followed by a long elimination phase. These results indicate low systemic bioavailability (F<0.05), similar to previous reports using male rats. Elimination pathways appeared to become saturated leading to delayed excretion after a single oral administration of the highest dose (1,000 mg/kg); no such saturation or delay was detected at lower doses. Chronic high exposures to TBBPA may result in competition for metabolism with endogenous substrates in extrahepatic tissues (e.g., SULT1E1 estrogen sulfation) resulting in endocrine disruption.

Comparison of 2,2-bis(bromomethyl)-1,3-propanediol induced genotoxicity in UROtsa cells and primary rat hepatocytes: relevance of metabolism and oxidative stress.

2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant used in urethane foams and polyester resins. In a two year dietary study, BMP caused neoplastic lesions at multiple sites including the urinary bladder of both rats and mice. However, liver was not a target tissue. We previously reported that BMP elicited oxidative DNA damage in a human uroepithelial cell line (UROtsa). The present in vitro study investigated the susceptibility of target (UROtsa cells) and non-target cells (primary rat hepatocytes) to BMP-induced genotoxicity. In contrast to hepatocytes, BMP exhibited greater genotoxic potential in UROtsa cells as evidenced by the concentration dependent increase in DNA strand breaks and DNA binding. Total content of intracellular GSH quantified in UROtsa cells (2.7±1.0nmol/mg protein) was 4 fold lower than that in hepatocytes (10.7±0.3nmol/mg protein). HPLC analysis indicated BMP was not metabolized and/or consumed in UROtsa cells at any of the concentrations tested (10-250µM) but was extensively converted to a mono-glucuronide in hepatocytes. These results demonstrate that a target cell line such as UROtsa cells are more susceptible to BMP-induced DNA damage when compared to non-target cells. This increased susceptibility may relate to the deficiency of antioxidant and/or metabolic capabilities in UROtsa cells.