Physiological effects of developmental exposure to flame retardant mixture Firemaster 550 or it's components

Firemaster 550 (FM550) is a flame retardant (FR) mixture which has become one of the most commonly used FRs in household items such as foam-based furniture and baby products. Because this mixture readily leaches from products, contamination of the environment and human tissues is widespread. Prior work by us and others has reported sex-specific behavioral deficits in rodents and zebrafish following early life exposure. In an effort to understand the mechanisms by which these behavioral effects occur, here we explored the effects of its constituents on behavioral outcomes previously shown to be altered by developmental FM550 exposure.  The FM550 commercial mixture is composed of two brominated compounds (BFR) and two organophosphate compounds (OPFRs) at almost equivalent proportions. Both the BFR and the OPFR components are differentially metabolized and structurally distinct, but similar to known neurotoxicants. Here we examined adult Wistar rat offspring socioemotional behaviors following perinatal exposure (oral, to the Dam) to vehicle, 2000 µg/day FM550, 1000 µg/day BFR or 1000 µg/day OPFR from gestation day 0 to weaning. Beginning on postnatal day 65 offspring from all groups were subjected to a series of behavioral tasks including open field, elevated plus maze, marble burying, social interaction tests, and running wheel.  Effects were exposure-, sex- and task-specific, with BFR exposure resulting in the most consistent behavioral deficits. Overall, exposed females showed more deficits compared to males across all dose groups and tasks. These findings help elucidate how different classes of flame retardants, independently and as a mixture, contribute to sex-specific behavioral effects of exposure.

Methods
Females were sacrificed on the day of estrus, and all rats were collected at approximately PND 247-261. Serum was collected from all rats via trunk blood. Wet weights were collected for adrenals, pituitaries, whole liver, kidneys, and hearts. The adrenals, pituitaries and median lobe of the livers was frozen on powdered dry ice and stored in -80°C. Using the ROUT outliers test (Q=1%), one BFR male and one OPFR female were identified and removed from adrenal and heart weight analysis, respectively. Final animal numbers were as follows: controls (14F, 14M); BFR (11F, 8M); OPFR (11F, 11M); and FM550 (14F, 15M).

Serum biochemistry was analyzed for 14 biochemical markers of general toxicity (listed in data set) to determine exposure effects. All procedures followed manufacturer’s protocols. On day of analysis, random samples of serum were thawed at 4°C and 120µL/sample added to the Comprehensive Diagnostic profile rotors (500–7123; Abaxis, Union City, CA). For quality assurance, a control sample (pooled from 20 random samples) was ran approximately every 25 samples. Rotors were placed in the VetScan VS2 Whole Blood chemistry analyzer and data exported. Final numbers were controls (13F,14M), BFR (11F,9M), OPFR (12F,11M) and FM550 (14F,15M). Although 21 statistical outliers from 6 biomarkers across female and male groups were identified, all data was used because no animal had greater than 2 outlying samples across all biomarkers.

Firemaster 550（FM550）是一种阻燃剂（flame retardant, FR）混合物，目前已成为泡沫家具、婴幼儿用品等家居用品中最常用的阻燃剂之一。由于该混合物极易从制品中浸出，已造成环境与人体组织的广泛污染。本团队及其他研究者此前已有报道称，啮齿类与斑马鱼在生命早期暴露后会出现性别特异性的行为缺陷。为阐明此类行为效应的发生机制，本研究探讨了其各组分对此前已被证实会因发育阶段暴露于FM550而发生改变的行为结局的影响。
商用FM550混合物由两种溴化阻燃剂（brominated flame retardants, BFR）与两种有机磷酸酯阻燃剂（organophosphate flame retardants, OPFRs）按近乎均等的比例复配而成。BFR与OPFR组分的代谢途径存在差异、结构各不相同，但均与已知的神经毒物具有相似性。本研究针对围产期（经口暴露于孕鼠）分别给予赋形剂、2000 μg/天FM550、1000 μg/天BFR或1000 μg/天OPFR（从妊娠第0天至断奶）的成年Wistar大鼠后代，检测其社会情感行为。自出生后第65天起，对所有分组的后代开展一系列行为学实验，包括旷场实验、高架十字迷宫实验、埋珠实验、社交互动实验以及跑轮实验。
研究发现，行为效应具有暴露方式、性别与实验任务特异性，其中BFR暴露引发的行为缺陷最为显著。整体而言，在所有剂量组与实验任务中，雌性暴露个体的行为缺陷程度均高于雄性。本研究结果有助于阐明不同类别阻燃剂单独及复配时，如何引发暴露后出现的性别特异性行为效应。

### 材料与方法
雌性大鼠于动情周期当日处死，所有大鼠的取材时间均为出生后第247~261天左右。通过断头采血收集所有大鼠的血清。采集肾上腺、垂体、全肝、肾脏与心脏的湿重。将肾上腺、垂体及肝中叶置于干冰粉末上速冻后保存于-80℃冰箱。采用ROUT离群值检验（Q=1%），分别在肾上腺重量分析与心脏重量分析中剔除1个BFR组雄性样本与1个OPFR组雌性样本。最终各组动物数量如下：对照组（雌性14只，雄性14只）；BFR组（雌性11只，雄性8只）；OPFR组（雌性11只，雄性11只）；FM550组（雌性14只，雄性15只）。
对血清进行生化分析，检测14项通用毒性生化标志物（详见数据集）以判断暴露产生的效应。所有操作均遵循制造商的实验方案。检测当日，血清样本于4℃解冻，每份样本取120 μL加入综合诊断分析转子（货号500–7123；Abaxis，美国加利福尼亚州尤宁城）。为保证质量控制，每约25个样本即插入1份混合对照样本（由20份随机样本混合而成）进行检测。将转子置于VetScan VS2全自动血液生化分析仪中运行，导出检测数据。最终各组样本数量如下：对照组（雌性13只，雄性14只）；BFR组（雌性11只，雄性9只）；OPFR组（雌性12只，雄性11只）；FM550组（雌性14只，雄性15只）。尽管在雌雄各组的6项生物标志物中共检出21个统计离群值，但由于无任何动物在所有生物标志物中存在超过2个异常样本，因此保留全部数据进行分析。