基于斑马鱼的等摩尔暴露下6PPD与6PPD-Q的神经与免疫毒性差异比较

Comparison on neurotoxicity and immunotoxicity between 6PPD and 6PPD-Q in zebrafish under equimolar exposure conditions

摘要:

N-(1,3-二甲基丁基)-N′-苯基对苯二胺[N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine, 6PPD]及其衍生物N-(1,3-二甲基丁基)-N′-苯基对苯醌[N-(1,3-dimethylbutyl)-N′-phenyl-p-benzoquinone, 6PPD-Q]已在环境中普遍检出，并对生态系统和人类健康构成潜在威胁。6PPD在环境和生物体内可等摩尔转化为6PPD-Q，但二者等摩尔暴露下的毒性差异及分子作用机制尚未明确。本研究在等摩尔条件下系统比较了二者的神经与免疫毒性。以斑马鱼为模式生物的实验结果显示，6PPD和6PPD-Q均诱导活性氧(reactive oxygen species, ROS)过量生成并抑制抗氧化酶表达，导致氧化损伤和免疫稳态失衡，同时激活先天免疫系统并引起免疫细胞增多。在神经发育方面，二者扰动神经功能基因表达，并诱发心包水肿、游囊闭合延迟和脊柱弯曲等畸形。值得注意的是，在环境相关浓度下，两者急性毒性相近，但在亚致死水平下，6PPD-Q表现出更强的毒性，其氧化损伤、免疫毒性、致畸性和神经毒性均较6PPD高约1-3倍，并显著削弱了幼鱼的感知与运动能力。实时荧光定量PCR (real-time quantitative PCR, RT-qPCR)结果表明，二者对神经和炎症相关基因的调控呈剂量依赖性。结合基因本体论(gene ontology, GO)、京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)、疾病本体论(disease ontology, DO)及核心基因分析，进一步揭示了6PPD与6PPD-Q在分子层次的作用焦点存在差异。本研究为识别和预警6PPD及6PPD-Q在环境与亚致死水平下的潜在风险提供了新的证据。

Abstract:

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and its derivative N-(1,3-dimethylbutyl)-N′-phenyl-p-benzoquinone (6PPD-Q) have been widely detected in the environment and pose potential threats to ecosystems and human health. Given that 6PPD can be equimolarly converted to 6PPD-Q in the environment and organisms, the toxicity differences and molecular mechanisms of action between the two under equimolar exposure remain unclear. This study systematically compared the neurotoxicity and immunotoxicity of the two substances under equimolar conditions. The results from the zebrafish model showed that both 6PPD and 6PPD-Q induced excessive production of reactive oxygen species (ROS) and inhibited the expression of antioxidant enzymes, leading to oxidative damage and immune dyshomeostasis. Meanwhile, they activated the innate immune system and caused an increase in immune cells. In terms of neural development, both disturbed the expression of neurofunctional genes and induced malformations such as pericardial edema, delayed swim bladder closure, and spinal curvature. Notably, at environmentally relevant concentrations, the two showed similar acute toxicity. However, at sublethal levels, 6PPD-Q exhibited stronger toxicity, with oxidative damage, immunotoxicity, teratogenicity, and neurotoxicity being 1-3 times higher than those of 6PPD, and it significantly impaired the sensory and motor abilities of larval fish. The results of real-time quantitative PCR (RT-qPCR) indicated that both substances regulated the expression of neuro- and inflammation-related genes in a dose-dependent pattern. The gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), disease ontology (DO), and hub gene analyses further revealed differences in the molecular-level action focuses between 6PPD and 6PPD-Q. This study provides new evidence for the identification and early warning of the potential risks of 6PPD and 6PPD-Q at environmental and sublethal levels.