Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada.
Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada.
Environ Pollut. 2022 Feb 15;295:118667. doi: 10.1016/j.envpol.2021.118667. Epub 2021 Dec 9.
Current approaches in chemical hazard assessment face significant challenges because they rely on live animal testing, which is time-consuming, expensive, and ethically questionable. These concerns serve as an impetus to develop new approach methodologies (NAMs) that do not rely on live animal tests. This study explored a molecular benchmark dose (BMD) approach using a 7-day embryo-larval fathead minnow (FHM) assay to derive transcriptomic points-of-departure (tPODs) to predict apical BMDs of fluoxetine (FLX), a highly prescribed and potent selective serotonin reuptake inhibitor frequently detected in surface waters. Fertilized FHM embryos were exposed to graded concentrations of FLX (confirmed at < LOD, 0.19, 0.74, 3.38, 10.2, 47.5 μg/L) for 32 days. Subsets of fish were subjected to omics and locomotor analyses at 7 days post-fertilization (dpf) and to histological and biometric measurements at 32 dpf. Enrichment analyses of transcriptomics and proteomics data revealed significant perturbations in gene sets associated with serotonergic and axonal functions. BMD analysis resulted in tPOD values of 0.56 μg/L (median of the 20 most sensitive gene-level BMDs), 5.0 μg/L (tenth percentile of all gene-level BMDs), 7.51 μg/L (mode of the first peak of all gene-level BMDs), and 5.66 μg/L (pathway-level BMD). These tPODs were protective of locomotor and reduced body weight effects (LOEC of 10.2 μg/L) observed in this study and were reflective of chronic apical BMDs of FLX reported in the literature. Furthermore, the distribution of gene-level BMDs followed a bimodal pattern, revealing disruption of sensitive neurotoxic pathways at low concentrations and metabolic pathway perturbations at higher concentrations. This is one of the first studies to derive protective tPODs for FLX using a short-term embryo assay at a life stage not considered to be a live animal under current legislations.
目前的化学危害评估方法面临着重大挑战，因为它们依赖于活体动物测试，这种方法既耗时、昂贵，又在伦理上存在争议。这些问题促使人们开发新的方法，这些方法不依赖于活体动物测试。本研究探索了一种使用 7 天胚胎-幼鱼法（FHM）的分子基准剂量（BMD）方法，以获得转录组起始点（tPOD），从而预测氟西汀（FLX）的顶极 BMD，FLX 是一种高度规定的、有效的选择性 5-羟色胺再摄取抑制剂，经常在地表水中检测到。受精的 FHM 胚胎暴露于不同浓度的 FLX（确认低于检测限、0.19、0.74、3.38、10.2、47.5μg/L）32 天。在受精后 7 天（dpf）对鱼类的一部分进行组学和运动分析，并在 32 dpf 时进行组织学和生物测量。转录组学和蛋白质组学数据的富集分析显示，与 5-羟色胺能和轴突功能相关的基因集受到显著干扰。BMD 分析得出 tPOD 值为 0.56μg/L（20 个最敏感的基因水平 BMD 的中位数）、5.0μg/L（所有基因水平 BMD 的第十个百分点）、7.51μg/L（所有基因水平 BMD 的第一个峰值的模式）和 5.66μg/L（途径水平 BMD）。这些 tPOD 可保护本研究中观察到的运动和体重减轻效应（10.2μg/L 的 LOEC），并反映了文献中报告的 FLX 的慢性顶极 BMD。此外，基因水平 BMD 的分布呈双峰模式，表明在低浓度下破坏了敏感的神经毒性途径，在高浓度下破坏了代谢途径。这是使用当前法规下不被视为活体动物的生命阶段的短期胚胎测定法为 FLX 得出保护 tPOD 的首批研究之一。
