Toxic Effects of Acute Benzo[a]pyrene Exposure on Blood Clam Tegillarca granosa and Its Potential Coping Mechanism

Abstract

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.

Keywords

• benzo[a]pyrene
• tegillarca granosa
• tissue damage
• oxidative stress
• neurotoxicity
• dna methylation