Increased perfluorooctanesulfonic acid (PFOS) toxicity and accumulation is associated with perturbed DHA and prostaglandin metabolism and increased OATP-transporter expression. Increased perfluorooctanesulfonic acid (PFOS) toxicity and accumulation is associated with perturbed DHA and prostaglandin metabolism and increased OATP-transporter expression

Perfluorooctanesulfonate (PFOS) is a widespread environmental pollutant with a long half-life and clearly negative outcomes on metabolic diseases such as fatty liver and diabetes. Male and female Cyp2b-null and humanized CYP2B6-transgenic (hCYP2B6-Tg) mice were treated with 0, 1, or 10 mg/kg/day PFOS for 21 days, and surprisingly it was found that PFOS was retained at greater concentrations in the serum and liver of hCYP2B6-Tg mice than Cyp2b-null mice with greater differences in the females. Thus, Cyp2b-null and hCYP2B6-Tg mice provide new models for investigating individual mechanisms for PFOS bioaccumulation and toxicity. Overt toxicity was greater in hCYP2B6-Tg mice (especially females) as measured by mortality; however, steato-sis occurred more readily in Cyp2b-null mice despite the lower PFOS liver concentrations. Tar-geted lipidomics and transcriptomics from PFOS treated Cyp2b-null and hCYP2B6-Tg mouse livers were performed and compared to PFOS retention and serum markers of toxicity by PCA. Several oxylipins, including prostaglandins, thromboxanes, and docosahexaenoic acid metabo-lites are associated or inversely associated with PFOS toxicity. Both lipidomics and tran-scriptomics indicate PFOS toxicity is associated with PPAR activity in all models. GO terms as-sociated with reduced steatosis were sexually dimorphic with lipid metabolism and transport increased in females and circadian rhythm associated genes increased in males. However, we can rule out that steatosis was initially protective from PFOS toxicity. Moreover, several transporters are associated with increased retention probably due to increased uptake. The strongest associa-tions are the organic anion transport proteins (Oatp1a4-6) genes and a long-chain fatty acid transport protein (fatp1), enriched in female hCYP2B6-Tg mice. PFOS uptake was also reduced in cultured murine hepatocytes by OATP inhibitors. The role of OATP1A6 and FATP1 in PFOS transport has not been tested. In summary, Cyp2b-null and hCYP2B6-Tg mice provided unique models for estimating the importance of novel mechanisms in PFOS retention and toxicity. Overall design: The goal of this experiment was to determine the gene expression differences between our Cyp2b-null and hCYP2B6-Tg mouse models that led to increased PFOS retention in the livers of hCYP2B6-Tg mice. The Cyp2b-null mouse model was generated via Crispr-Cas9 knockout, and the hCYP2B6-Tg mouse was generated on the null background by breeding it with a CYP2B6/2A13/2F1-transgenic mouse (Wei, 2012). Male and female Cyp2b-null and hCYP2B6-Tg mice were treated with 0, 1, or 10 mg/kg/day PFOS, and greater PFOS retention was observed in the hCYP2B6-Tg mice livers. Total RNA was then extracted from 4 mouse livers in each group for RNA-seq. Differential gene expression analysis was performed to compare expression profiles betwen PFOS treatments within a genotype and across genotypes at each particular PFOS concentration.

全氟辛烷磺酸（Perfluorooctanesulfonate, PFOS）是一类分布广泛的环境污染物，具有较长的生物半衰期，且对脂肪肝、糖尿病等代谢性疾病具有明确的不良影响。本研究对Cyp2b基因敲除（Cyp2b-null）及人源化CYP2B6转基因（hCYP2B6-Tg）雌雄小鼠分别以0、1或10 mg/kg/天的剂量暴露PFOS 21天，意外发现hCYP2B6-Tg小鼠血清与肝脏中的PFOS浓度显著高于Cyp2b-null小鼠，且雌性小鼠的浓度差异更为显著。因此，Cyp2b-null与hCYP2B6-Tg小鼠可为探究PFOS生物富集与毒性的个体差异机制提供全新的动物模型。
以死亡率为评价指标时，hCYP2B6-Tg小鼠（尤其是雌性个体）的显性毒性更为显著；然而尽管Cyp2b-null小鼠肝脏中的PFOS浓度更低，其脂肪肝的发生率却更高。本研究对PFOS暴露后的Cyp2b-null与hCYP2B6-Tg小鼠肝脏样本开展了靶向脂质组学与转录组学分析，并通过主成分分析（Principal Component Analysis, PCA）将组学数据与PFOS蓄积水平及血清毒性标志物进行关联分析。
多种氧脂素（oxylipins）——包括前列腺素（prostaglandins）、血栓素（thromboxanes）及二十二碳六烯酸（docosahexaenoic acid）代谢物——与PFOS毒性呈正相关或负相关。脂质组学与转录组学分析均表明，在所有模型中，PFOS毒性均与过氧化物酶体增殖物激活受体（Peroxisome Proliferator-Activated Receptor, PPAR）的活性相关。
与脂肪肝减轻相关的基因本体（Gene Ontology, GO）术语存在性别二态性：雌性小鼠中脂质代谢与转运相关基因表达上调，雄性小鼠中则为昼夜节律相关基因表达升高。但本研究可排除“脂肪肝最初可对PFOS毒性起到保护作用”这一假说。
此外，多种转运蛋白与PFOS蓄积增加相关，这可能与PFOS摄取增强有关。其中关联最强的是有机阴离子转运多肽1a4-6（organic anion transport protein 1a4-6, Oatp1a4-6）基因及长链脂肪酸转运蛋白1（fatty acid transport protein 1, fatp1），这些基因在雌性hCYP2B6-Tg小鼠中富集显著。在培养的小鼠肝细胞（murine hepatocytes）中，OATP抑制剂可降低PFOS的摄取。目前尚未验证OATP1A6与FATP1在PFOS转运中的具体作用。
综上，Cyp2b-null与hCYP2B6-Tg小鼠为阐明PFOS蓄积与毒性的新型机制提供了独特的动物模型。
整体实验设计：本实验旨在明确两种小鼠模型（Cyp2b-null与hCYP2B6-Tg）中导致hCYP2B6-Tg小鼠肝脏PFOS蓄积增加的基因表达差异。Cyp2b-null小鼠通过CRISPR-Cas9基因编辑技术构建；hCYP2B6-Tg小鼠则以Cyp2b-null小鼠为背景，通过与CYP2B6/2A13/2F1转基因小鼠杂交获得（Wei等，2012）。将Cyp2b-null与hCYP2B6-Tg雌雄小鼠分别以0、1或10 mg/kg/天的剂量暴露PFOS，证实hCYP2B6-Tg小鼠肝脏中PFOS蓄积水平更高。随后从每组4只小鼠的肝脏中提取总RNA进行RNA测序（RNA-sequencing, RNA-seq）。针对不同基因型在相同PFOS暴露浓度下，以及同一基因型在不同PFOS暴露剂量下的基因表达谱，分别开展差异表达基因分析。