苯并[a]芘(BaP)急性暴露对泥蚶消化腺的毒性效应及其应对机制初探

Toxic effects of acute benzo[a]pyrene exposure on blood clam Tegillarca granosa and its potential coping mechanism

中文关键词: 苯并[a]芘  泥蚶  组织损伤  氧化应激  神经毒性  DNA甲基化

英文关键词: Benzo[a]pyrene  Tegillarca granosa  Tissue damage  Oxidative stress  Neurotoxicity  DNA methylation

中文摘要:

      本文研究了苯并[a]芘(benzo[a]pyrene, BaP)急性暴露对泥蚶(Tegillarca granosa)的毒性效应，并以泥蚶的消化腺为目标组织，对泥蚶抵抗BaP毒性的机制进行了初步探究。对样本进行HE染色并对所采集的图像进行分析处理，结果发现，暴露于BaP导致泥蚶消化腺组织出现坏死。对样本进行脂质过氧化并进行DNA氧化损伤测定的结果显示，暴露于BaP导致泥蚶消化腺氧化压力增加，进而导致脂质过氧化以及DNA氧化损伤等细胞层面的损伤。抗氧化酶活性测定结果显示，BaP胁迫诱导泥蚶产生氧化应激反应，抗氧化防御体系积极参与BaP诱导的氧化应激调控过程。神经毒性指标测定结果显示，BaP胁迫可能影响泥蚶神经冲动的传导而具有神经毒性。此外，DNA甲基化水平测定及抗氧化酶基因表达测定的结果显示，泥蚶可能通过降低DNA甲基化水平来激活抗氧化系统以对抗BaP毒性。综上所述，急性BaP暴露会对泥蚶产生显著的毒性效应，以组织损伤、氧化应激反应和神经毒性为主要表征；泥蚶可能通过调整自身DNA甲基化水平来对抗BaP毒性效应。通过本研究，有望为深入探索双壳贝类应对石油污染物胁迫的内在调控机制提供新思路，同时为石油污染威胁下泥蚶的生物资源保护提供参考。

英文摘要:

      Spilled petroleum pollution caused by seaborne oil transportation is a major marine environmental problem in the world. Petroleum pollutants contain significant amounts of low-molecular-weight alkanes and aromatics that induce toxic effects once taken up by marine organisms. Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds with the most significant toxic environmental effects. As bivalves, which have a strong enrichment and tolerance capacity for PAHs in water, are widely distributed and easy to obtain, they are often used as model organisms for monitoring and evaluating offshore marine pollution. The blood clam (Tegillarca granosa), a bivalve with high economic value, is widely distributed in mudflats along the coast of Zhejiang Province. Blood clams have characteristics of benthic life, making them more likely to be exposed to petroleum pollutants. At present, there are few studies on the toxic effects of PAH exposure on blood clams. Herein, we chose benzo[a]pyrene (BaP), a typical PAH congener, as a contaminant, and investigated the toxic effects of acute BaP exposure on blood clams and their potential coping mechanisms. Blood clams required for the experiment were collected from Dongji Island and acclimated for a week in the laboratory. After acclimation, several healthy blood clams were randomly divided into the artificial sea water (ASW) (control group), dimethyl sulfoxide (DMSO) (solvent control group, 0.01% VDMSO/VASW), 10 μg/L BaP exposure group, and 100 μg/L BaP exposure groups based on the previous studies of our research group combined with the literature. Each group was set up with three replicates, each containing 40 individuals. The experiment lasted 96 h, and separate glass tanks were used to place all individuals in each replicate. One blood clam was randomly selected from each replicate of each concentration group at 0, 24, 48, and 96 h of exposure. After dissection on ice, the digestive gland was immediately separated using sterile forceps and scissors and stored at –80℃. The paraffin section was then used to observe the lesions of the digestive gland. A colorimetric assay was used to determine the activities of antioxidant enzymes and key enzymes involved in neurotransmission at different time points. The cell damage degree was also determined by measuring the levels of 8-hydroxy-2′- deoxyguanosine (8-OHdG) and malondialdehyde (MDA). In addition, enzyme-linked immunosorbent assays were used to determine DNA methylation levels. Finally, we measured the relative mRNA expression of antioxidant enzymes and performed a correlation analysis. Results showed prominent hemocyte infiltration and necrotic areas in the digestive gland of the blood clam after a total of 96 h of exposure to 10 and 100 μg/L BaP, indicating inflammation. The sloughing of digestive cells from the inner wall of the digestive tubule led to atrophy. Increased oxidative stress was indicated by elevated MDA and 8-OHdG content, leading to damage at the cellular level, such as lipid peroxidation and oxidative DNA damage. The activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione-S-transferase (GST), increased significantly 24 h post-exposure. This phenomenon further indicated that BaP exposure induced oxidative stress in the blood clam, with the antioxidant defense system actively regulating BaP-induced oxidative stress. The activities of two key neurotransmitter enzymes, acetylcholinesterase and acetylcholine transferase, were significantly reduced, indicating that the stress caused by BaP may induce neurotoxicity in the blood clam. In addition, we analyzed the changes in DNA methylation levels and gene expression of antioxidant enzymes in the blood clam under acute BaP exposure, finding that the DNA methylation levels were significantly decreased compared to pre-exposure, while the mRNA expression of SOD, CAT, and GST was significantly increased. Correlation analysis showed a negative correlation between DNA methylation and the gene expression of antioxidant enzymes, implying that blood clams may activate the antioxidant systems to fight against BaP toxicity by reducing DNA methylation levels. In conclusion, acute BaP exposure exerts a significant toxic effect on the blood clam, primarily characterized by histological damage, oxidative stress, and neurotoxicity. Moreover, changes in DNA methylation levels in the blood clam may be involved in the regulatory process of BaP toxic effects. This study is expected to provide new ideas for the in-depth exploration of the intrinsic regulatory mechanisms of bivalves in response to petroleum pollutant stress, and will be beneficial for resource conservation of the blood clam under the threat of petroleum pollution.