Developmental Exposure to the Flame Retardant Mixture Firemaster 550 Compromises Adult Bone Integrity in Male but not Female Rats.

Flame retardants (FRs) are used in a variety of common items from furniture to carpet to electronics to reduce flammability and combustion, but the potential toxicity of these compounds is raising health concerns globally. Organophosphate FRs (OPFRs) are becoming more prevalent as older, brominated FRs are phased out, but the toxicity of these compounds, and the FR mixtures that contain them, is poorly understood. Work in a variety of in vitro model systems has suggested that FRs may induce metabolic reprogramming such that bone density is compromised at the expense of increasing adiposity. To address this hypothesis, the present studies maternally exposed Wistar rat dams orally across gestation and lactation to 1000 µg daily of the FR mixture Firemaster 550 (FM 550) which contains a mixture of brominated FRs and OPFRs. At six months of age, the offspring of both sexes were examined for evidence of compromised bone composition. Bone density, composition, and marrow were all significantly affected, but only in males. The fact that the phenotype was observed months after exposure suggests that FM 550 altered some fundamental aspect of mesenchymal stem cell reprogramming. The severity of the phenotype and the human-relevance of the dose employed, affirm this is an adverse outcome meriting further exploration.


Introduction
Chemical flame retardants (FRs) are commonly applied to polymers, foam, resins, electronics and construction materials to reduce their flammability or delay their combustion. Because they are not chemically bound, they readily escape and accumulate in indoor dust. Over the past few decades, it has become increasingly apparent that some of these FRs have toxic properties. The FR mixture Firemaster ® 550 (FM 550) entered commercial use in the early 2000s and rapidly became one of the most commonly detected FRs in polyurethane foam used and sold in the US [1][2][3][4]. FM 550 contains a mixture of organophosphates: (1) triphenyl phosphate (TPHP; previously abbreviated TPP); and (2) a mixture of isopropylated triphenylphosphate isomers (ITPs); as well as two brominated compounds (3)
Histomorphometric analyses of the femurs are shown in Figures 3 and 4. In the hematoxylin and eosin staining, the number osteocytes (F1,12 = 8.72; p < 0.05) and percentage of trabecular bone (F1,12 = 7.75; p < 0.05) were both significantly decreased in FM 550-exposed males. FM 550 also markedly increased the percentage of yellow bone marrow in male rats (F1,12 = 8.49; p < 0.05; Figure 3). Masson's trichrome staining revealed that FM 550 decreased the number osteoblasts in the femur (F1,11 = 5.26; p < 0.05; Figure 4A  . Two-way ANOVA followed by the Holm-Sidak post hoc test. (n = 4 per group). * p < 0.05 different from control of the same sex, ** p < 0.01 different from control of the same sex, # p < 0.05 different from exposed females, ### p < 0.001 different from exposed females. . Two-way ANOVA followed by the Holm-Sidak post hoc test. (n = 4 per group). * p < 0.05 different from control of the same sex, ** p < 0.01 different from control of the same sex, # p < 0.05 different from exposed females, ### p < 0.001 different from exposed females.

Discussion
Our present findings reveal, for the first time in vivo, that developmental exposure to the flame retardant mixture FM 550 can compromise bone health in adult male rats long after exposure ceases. This phenomenon, in combination with significantly elevated body weight and left ventricle thickness observed in a prior, similar, study [6], suggests that overall metabolic health may be impaired. These data are consistent with numerous in vitro studies suggesting that FM 550, its

Discussion
Our present findings reveal, for the first time in vivo, that developmental exposure to the flame retardant mixture FM 550 can compromise bone health in adult male rats long after exposure ceases. This phenomenon, in combination with significantly elevated body weight and left ventricle thickness observed in a prior, similar, study [6], suggests that overall metabolic health may be impaired. These data are consistent with numerous in vitro studies suggesting that FM 550, its components, and structurally related FRs, heighten adiposity at the expense of bone formation [10,[31][32][33][34][35][36][37][38][39][40][41][42][43].
Bone marrow mesenchymal stem/stromal cells (BMSCs) have multipotential to differentiate into osteoblasts, adipocytes, chondrocytes or myoblasts. Molecular factors and differing signaling pathways controlling transcription may alternatively trigger events that direct BMSCs to osteogenesis or adipogenesis [34]. In cases where BMSCs are not directed towards osteogenesis, bone formation may be impaired resulting in deficient bone microarchitecture. In our results, bone formation was deficient in FM550-exposed males, as demonstrated by the microCT analysis. This supports the hypothesis that early exposure to FM 550 attenuated bone differentiation in favor of adipocyte differentiation, which is suggested by the decrease in trabecular bone and the number of osteoblasts in opposition to the increase in yellow bone marrow percentage.
Because it is a mixture, and the components are rapidly metabolized to several different metabolites [2], it is not possible from this single study to ascertain which of the parent compounds or metabolites may have contributed to the observed effects. Data from a range of experimental systems, however, provide compelling evidence that the organophosphate components of FM 550 are likely the primary drivers of this phenotype. Experiments in murine 3T3-L1 preadipocytes revealed that FM550 and its components TPHP, IPTP, and TBPH, but not TBB, dose-dependently induces lipid accumulation as well as PPARγ-mediated adipocyte protein 2 (aP2) enhancer activity [35]. This upregulation was blocked by the PPARγ-selective antagonist GW9662. Chromatin immunoprecipitation further confirmed a role for PPARγ by revealing that IPTP and TPP induced recruitment of PPARγ to the regulatory region of aP2. Work in ex vivo fetal murine limb bud cultures also identified TPHP as particularly detrimental to bone formation via suppression of Sox9, Runx2 and Sp7; genes known to be regulated by PPARγ [31]. Similarly, FM 550, and its organophosphate components, induce adipogenesis in human primary preadipocytes via a mechanism that highly implicates PPARγ [35]. A recent study by the National Toxicology Program (NTP), identified TPHP as a "high priority" chemical meriting additional testing due to evidence of developmental and neurotoxic properties comparable to the brominated flame retardants (largely polybrominated diphenyl ethers or PBDEs) it was meant to replace [36]. While bone density and composition were not mentioned specifically, it cautions that modern FRs are structurally similar to, and likely just as toxic, as the chemicals they replaced.
Notably, the present study is also consistent with prior work linking organophosphate pesticides with disrupted bone formation and maintenance [37]. Organophosphate pesticide studies may yield additional insight as to possible mechanisms of organophosphate FR action. For example, chlorpyrifos in combination with retinoic acid (RA), directs commitment of mesenchymal stem cells to adipogenic differentiation through a process involving a crosstalk at GSK3β signaling. Similarly, simultaneous subchronic oral exposure to selenium (5 mg) and diazinon (40 mg) in drinking water for 90 days induced changes in macroscopic and microscopic structures of adult male rat femurs including decreased femoral length and cortical bone thickness [38]. Because they contain acetylcholinesterase, MSC regulation and differentiation is thought to be disrupted by organophosphate pesticides via interference with acetylcholine signaling [39]. A role for sex has not been considered in much of the pesticide work, but should be a factor in future studies with FRs.
Importantly, an adverse role for the brominated compounds in the FM 550 FR mixture cannot be ruled out. Notably, two EPA-mandated studies conducted by Chemtura using only the brominated components of FM 550 (a two generation reproductive study (MPI Research Study 1038-008; "CN-2065: An Oral Two-Generation Reproduction and Fertility Study in Rats") and a developmental toxicity study (MPI Research Study 1038-006; CN-2065:Prenatal Developmental Toxicity Study in Rats") also found bone-related phenotypes, albeit at much higher doses. Exposures in both studies ranged from 15 to 300 mg/kg/day via oral gavage, which is typical for a guideline study conducted for regulatory purposes. Observations included fused cervical vertebral neural arches in fetuses of exposed dams, an effect attributed to exposure at the 300 mg/kg dose but classified as "incidental" at the 100 mg/kg dose. In both studies, the no observable adverse effects level (NOAEL) was set at 50 mg/kg/day, a level far exceeding typical human exposures and the dose used in the present study.

Materials and Methods
All tissues were obtained from animals used for a prior study, the detailed methods of which are published elsewhere [8] using the ARRIVE guidelines checklist (http://www.nc3rs.org.uk/ARRIVE). That study used three orally administered doses of FM 550. Femoral samples from the highest dose group (1000 µg per day;~3.3 mg/kg bw to the dam) and sex-matched vehicle (ethanol) controls were used for the present study. Accuracy of FM 550 dosing solution was confirmed by gas chromatography mass spectrometry. All dosing and testing was done blinded to exposure group.
Animal care and maintenance were conducted in accordance with the applicable portions of the Animal Welfare Act and the U.S. Department of Health and Human Services' "Guide for the Care and Use of Laboratory Animals" and approved by the North Carolina State University (NCSU) Institutional Animal Care and Use Committee (protocol ID# 17-058-B under Animal Welfare Assurance #D16-00214). All animals were obtained from an existing colony of Wistar rats maintained in a reversed light environment that minimizes exogenous exposure to endocrine disrupting compounds (EDCs) at the NCSU Biological Resource Facility. Study animals were kept in a climate controlled room at 25 • C and 45-60% average relative humidity on a phytoestrogen free diet (Teklad 2020, Harlan), and housed in thoroughly washed polysulfone cages with glass water bottles (rubber stoppers and metal sippers) and woodchip bedding to minimize unintentional exposure to endocrine disrupting compounds [40,41].
Daily oral exposure of the dams (n = 8 in the control group and n = 7 in the 1000 µg FM 550 group because one dam failed to litter) spanned gestational day (GD) 8 through weaning (postnatal day (PND) 21) by dispensing 20 µL of ethanol-based solution containing the appropriate dose onto 1 4 of a soy-free food treat pellet (apple or chocolate flavored AIN-76A Rodent Diet Test Tabs, Test Diet, Richmond, IN, USA). Oral dosing via food treats models human exposure while minimizing handling stress [42][43][44]. Litters were standardized to 10 (5:5 sex ratio whenever possible) on PND 1. On PND 21 pups were weaned into same sex littermate groups of up to 3 and housed in the same conditions as the dams. Animals were euthanized as adults (6 months of age), weighed, and the tissues distributed and archived for a variety of studies (detailed in Baldwin et al. [8]). Hind legs were surgically removed from a subset of animals at the time of euthanasia, frozen on dry ice and stored at −80 • C. Whole legs (n = 4 per sex per exposure group; one per sex per litter) were shipped to the Macari lab on dry ice for bone analyses.

Bone Histomorphometry
Measurements were performed at the distal femur excluding the growth plate as previously described [45]. The femurs were fixed in 10% formalin, decalcified in 14% EDTA and embedded in paraffin. Sagittal sections of 5 µm were stained using hematoxylin and eosin for density of osteocyte counting per trabecular bone area, tartrate resistant acid phosphatase (TRAP; Sigma-Aldrich, Saint Louis, MO, USA) for osteoclast TRAP positive cells analysis, and Masson's trichrome staining for osteoblast counting per trabecular bone perimeter. The percentages of red bone marrow, yellow bone marrow and trabecular number were determined at 40× magnification using an ocular micrometer containing a 121-point grid. Five sections per animal were evaluated under microscope (Olympus AX70 Light Microscope, Olympus Corporation, Tokyo, Japan).

Statistical Analysis
The litter was the statistical unit (one animal per sex per litter was used). Data are presented as the mean ± standard error of the mean (SEM). Body weight data were compared within sex by Student t-test. For bone data, differences among groups were analyzed by two-way ANOVA followed by the Holm-Sidak post hoc test. In all cases, p ≤ 0.05 was considered statistically significant.

Conclusions
Here we show, for the first time in vivo, that developmental exposure to FM 550, which contains TPHP and other suspected toxicants, adversely impacts bone health in adult males. The fact that the phenotype was observed months after exposure suggests that FM 550 altered some fundamental aspect of mesenchymal stem cell reprogramming, but this requires confirmation. While a small study with limited numbers, given the severity of the phenotype, use of different methodologies for bone analysis, consistency with prior in vitro data in multiple species, and the human-relevance of the dose employed, these data merit follow-up work to further explore this potential adverse outcome. Mode of action and possible methods of ameliorating this effect are of primary interest.