Locomotor ability and brain gene expression in Drosophila melanogaster exhibit non-monotonic dose-response to HFPO-DA

Per- and polyfluoroalkyl substances (PFAS) are persistent pollutants known for their bio-accumulative properties and prevalence in water supplies and household products. Although legacy PFAS, such as perfluorooctanoic acid, are phased out in the U.S. due to public health concerns, a PFAS variant hexafluoropropylene oxide-dimer acid (HFPO-DA) is an emerging replacement. HFPO-DA is a potential neurotoxicant that has been shown to cause dopaminergic neurodegeneration. We investigated the bioaccumulative potential of HFPO-DA and its effects on lifespan, locomotor activity, and brain gene expression in female and male Drosophila melanogaster (fruit flies). Flies were collected less than 4 hours post-eclosion and exposed to 0, 10, 10^2, 10^3, or 10^4 mg/kg/day HFPO-DA. To measure the effect of HFPO-DA on lifespan, surviving flies from each exposure were recorded every 24 hours. Flies were subjected to a negative geotaxis assay at 3, 7, and 14 days of exposure to measure the effects of acute, sub-chronic, and chronic exposures on locomotor ability. To capture HFPO-DA-induced sexually dimorphic gene expression responses in the brain, we sequenced brain-specific mRNA from flies exposed for 3, 7, or 14 days. The Bioconcentration Factor was 0.031 for females, and 0.026 for males. Dose and median lifespan were negatively correlated in both female (R^2 adj = 0.77, p<0.0001) and male flies (R^2 adj = 0.77, p<0.0001). Log-rank Mantel-Cox tests and one-way ANOVAs revealed that median lifespan was reduced in females starting at 10 mg/kg/day and in males starting at 10^2 mg/kg/day (p< 0.01). Acute exposure at 10 mg/kg/day significantly decreased locomotor ability in females (p<0.0001) while acute exposures at 1 mg/kg/day decreased locomotor activity in males (p <0.0001). Among 7500 genes analyzed, pairwise gene expression comparison between controls and treatments identified 2496 differentially expressed genes. While HFPO-DA does not readily bioaccumulate in fruit fly bodies, high-dose exposures have sex-specific effects on lifespan, locomotor ability, and brain gene expression. LOAEL for median lifespan is 10 mg/kg/day in females, and 10^2 mg/kg/day in males. Both locomotor ability and brain gene expression exhibited non-conventional dose-response as seen in other endocrine disrupting chemical exposures. Overall design: Gene expression profiling analysis of shallow RNA-seq data for D. melanogaster 3, 7, and 14 day exposures to hexafluoropropylene oxide dimer acid (HFPO-DA); three replicates of 24 experimental conditions (2 sex, 3 different dosages of HFPO-DA plus a control, 3 exposure durations) were generated, with each condition containing around 100 fly brains.

全氟和多氟烷基物质（Per- and polyfluoroalkyl substances, PFAS）是一类持久性污染物，以生物累积特性以及在供水系统和家居产品中的广泛分布而闻名。尽管全氟辛酸（perfluorooctanoic acid）等传统PFAS因公共健康顾虑已在美国逐步淘汰，但新型PFAS变体六氟丙烯氧化物二聚酸（hexafluoropropylene oxide-dimer acid, HFPO-DA）正成为其替代物。HFPO-DA是一种潜在神经毒物，已被证实可引发多巴胺能神经元变性。本研究调查了HFPO-DA的生物累积潜力，及其对雌雄黑腹果蝇（Drosophila melanogaster，即果蝇）寿命、运动活性与脑部基因表达的影响。
实验中，研究人员于果蝇羽化后4小时内收集样本，将其分别暴露于0、10、10²、10³、10⁴ mg/kg/天的HFPO-DA环境中。为评估HFPO-DA对果蝇寿命的影响，每24小时记录各暴露组的存活果蝇数量。在暴露第3、7、14天时分别开展负趋地性试验，以检测急性、亚慢性与慢性暴露对果蝇运动能力的影响。为捕捉HFPO-DA诱导的脑部性别二态性基因表达响应，研究人员对暴露3、7或14天的果蝇的脑部特异性mRNA进行了测序。
研究结果显示：雌性果蝇的生物浓缩因子（Bioconcentration Factor, BCF）为0.031，雄性为0.026；雌性与雄性果蝇的暴露剂量与中位寿命均呈负相关（校正R²=0.77，p<0.0001）。对数秩曼特尔-考克斯检验（Log-rank Mantel-Cox tests）与单因素方差分析（one-way ANOVAs）结果表明，雌性果蝇的中位寿命自10 mg/kg/天剂量起显著降低，雄性则自10² mg/kg/天剂量起出现显著降低（p<0.01）。10 mg/kg/天的急性暴露可显著降低雌性果蝇的运动能力（p<0.0001），而1 mg/kg/天的急性暴露则会降低雄性果蝇的运动活性（p<0.0001）。在分析的7500个基因中，对照组与处理组的成对基因表达比较共鉴定出2496个差异表达基因。尽管HFPO-DA在果蝇体内不易发生生物累积，但高剂量暴露会对果蝇的寿命、运动能力与脑部基因表达产生性别特异性影响。雌性果蝇中位寿命的最低可见有害作用水平（Lowest Observed Adverse Effect Level, LOAEL）为10 mg/kg/天，雄性为10² mg/kg/天。与其他内分泌干扰物暴露的情况类似，果蝇的运动能力与脑部基因表达均表现出非常规的剂量-反应关系。
本研究的整体实验设计为：对暴露于六氟丙烯氧化物二聚酸（HFPO-DA）3、7、14天的黑腹果蝇开展浅层RNA测序（shallow RNA-seq）的基因表达谱分析；共设置24种实验条件（2种性别、3种HFPO-DA剂量组加1个对照组、3种暴露时长），每种条件设置3个生物学重复，每个重复包含约100个果蝇脑部组织。