The immunotoxicity mechanism of hemocytes in Chlamys farreri incubated with noradrenaline and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide alone or in combination.

Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) is the active intermediate metabolite of benzo[a]pyrene (B[a]P) and is considered the ultimate immunotoxicant. The neuroendocrine immunoregulatory network of bivalves is affected under pollutant stress. Besides, bivalves are frequently affected by pollutants in marine environments, yet the combined effects of neuroendocrine factors and detoxification metabolites on bivalves under pollutant stress and the signal pathways that mediate this immunoregulation are not well understood. Therefore, we incubated the hemocytes of Chlamys farreri with the neuroendocrine factor noradrenaline (NA) and the B[a]P detoxification metabolite BPDE, alone or in combination, to examine the immunotoxic effects of NA and BPDE on the hemocytes in C. farreri. Furthermore, the effects of NA and BPDE on the hemocyte signal transduction pathway were investigated by assessing potential downstream targets. The results revealed that NA and BPDE, alone or in combination, resulted in a significant decrease in phagocytic activity, bacteriolytic activity and the total hemocyte count. In addition, the immunotoxicity induced by BPDE was further exacerbated by co-treatment with NA, and the two showed synergistic effects. Analysis of signaling pathway factors showed that NA activated G proteins by binding to α-AR, which transmitted information to the Ca2+-NF-κB signaling pathway to regulate the expression of phagocytosis-associated proteins and regulated cytokinesis through the cAMP signaling pathway. BPDE could activate PTK and affect phagocytosis and cytotoxicity proteins through Ca2+-NF-κB signal pathway, also affect the regulation of phagocytosis and cytotoxicity by inhibiting the AC-cAMP-PKA pathway to down-regulate the expression of NF-κB and CREB. In addition, BPDE and NA may affect the immunity of hemocytes by down-regulating phagocytosis-related proteins through inhibition of the lectin pathway, while regulating the expression of cytotoxicity-related proteins through the C-type lectin. In summary, immune parameters were suppressed through Ca2+ and cAMP dependent pathways exposed to BPDE and the immunosuppressive effects were enhanced by the neuroendocrine factor NA.

Potential pathway and mechanisms underlining the immunotoxicity of benzo[a]pyrene to Chlamys farreri.

The long-distance migration of polycyclic aromatic hydrocarbons (PAHs) promotes their release into the marine environment, posing a serious threat to marine life. Studies have shown that PAHs have significant immunotoxicity effects on bivalves, but the exact mechanism of immunotoxicity remains unclear. This paper aims to investigate the effects of exposure to 0.4, 2, and 10 µg/L of benzo(a)pyrene (B[a]P) on the immunity of Chlamys farreri under environmental conditions, as well as the potential molecular mechanism. Multiple biomarkers, including phagocytosis rate, metabolites, neurotoxicity, oxidative stress, DNA damage, and apoptosis, were adopted to assess these effects. After exposure to 0.4, 2, and 10 µg/L B[a]P, obvious concentration-dependent immunotoxicity was observed, indicated by a decrease in the hemocyte index (total hemocyte count, phagocytosis rate, antibacterial and bacteriolytic activity). Analysis of the detoxification metabolic system in C. farreri revealed that B[a]P produced B[a]P-7,8-diol-9,10-epoxide (BPDE) through metabolism, which led to an increase in the expression of protein tyrosine kinase (PTK). In addition, the increased content of neurotransmitters (including acetylcholine, γ -aminobutyric acid, enkephalin, norepinephrine, dopamine, and serotonin) and related receptors implied that B[a]P might affect immunity through neuroendocrine system. The changes in signal pathway factors involved in immune regulation indicated that B[a]P interfered with Ca2+ and cAMP signal transduction via the BPDE-PTK pathway or neuroendocrine pathway, resulting in immunosuppression. Additionally, B[a]P induced the increase in reactive oxygen species (ROS) content and DNA damage, as well as an upregulation of key genes in the mitochondrial pathway and death receptor pathway, leading to the increase of apoptosis rate. Taken together, this study comprehensively investigated the detoxification metabolic system, neuroendocrine system, and cell apoptosis to explore the toxic mechanism of bivalves under B[a]P stress.

Immunotoxicity pathway and mechanism of benzo[a]pyrene on hemocytes of Chlamys farreri in vitro.

Benzo[a]pyrene (B[a]P), a typical PAHs widely existing in the marine environment, has been extensively studied for its immunotoxicity due to its persistence and high toxicity. Nevertheless, the immunotoxicity mechanism remain incompletely understood. In this study, isolated hemocytes of Chlamys farreri were exposed at three concentrations of B[a]P (5, 10 and 15 µg/mL), and the effects of B[a]P on detoxification metabolism, signal transduction, humoral immune factors, exocytosis and phagocytosis relevant proteins and immune function at 0, 6, 12, 24 h were studied. Results illustrated the AhR, ARNT and CYP1A1 were significantly induced by B[a]P at 12 h. Additionally, the content of B[a]P metabolite BPDE increased in a dose-dependent manner with pollutants. Under B[a]P stimulation, the expressions of PTK (Src, Fyn) and PLC-Ca2+-PKC pathway gene increased significantly, while the transcription level of AC-cAMP-PKA pathway gene decreased remarkably. Additionally, the expressions of nuclear transcription factors (CREB, NF-κB), complement system genes and C-type lectin genes up-regulated obviously. The gene expressions of phagocytosis and exocytosis related proteins were also notably affected. 5 µg/mL B[a]P could promote phagocytosis in a transitory time, but with the increase of exposure time and concentration of B[a]P, the phagocytosis, antibacterial and bacteriolytic activities gradually decreased. These results indicated that similar to vertebrates, BPDE, the metabolite of B[a]P, mediated downstream signal transduction via PTK in bivalves. The declined of the immune defense ability of hemocytes might be closely related to the inhibition of AC-cAMP-PKA pathway and the imbalance of intracellular Ca2+ pathway. In addition, the results manifested that complement and lectin systems play a significant role in regulating immune response. In this study, the direct relationship between detoxification metabolism and immune signal transduction in bivalves under B[a]P stress was demonstrated for the first time, which provided important information for the potential molecular mechanism of B[a]P-induced immune system disorder in bivalves.

The mechanism of apoptosis of Chlamys farreri hemocytes under benzopyrene stress in vitro.

Hemocytes are critical to the immune defense system of bivalves, and polycyclic aromatic hydrocarbons (PAHs) can mediate the immunity of bivalves by affecting the apoptosis of hemocytes. However, the underlying mechanism is still unclear. Chlamys farreri, as an important economic bivalve, was selected as the research subject for this experimentation. The hemocytes were exposed to typical PAHs-benzopyrene (B[a]P) in vitro to explore the apoptosis mechanism through detecting oxidative stress and oxidative damage-related indicators, apoptosis pathway factors, and apoptosis rate within 24 h. The results showed that the reactive oxygen species (ROS) and benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) content in hemocytes increased significantly under B[a]P exposure, while antioxidant genes, glutathione peroxidase content and total antioxidant capacity all showed a trend of first rising and subsequent falling. B[a]P also caused serious damage to DNA and lysosomal membrane stability. The proapoptotic factors genes in the mitochondrial apoptosis pathway were significantly up-regulated, and the anti-apoptotic gene Bcl-2 was significantly down-regulated. Besides, mitochondrial membrane potential stability was significantly reduced and caspase 9 enzyme activity was significantly improved with the B[a]P stimulation. The factors of death receptor pathway were also significantly up-regulated by B[a]P. Moreover, the expression levels of Mitogen-Activated Protein Kinases were also induced. The gene expression and enzyme activity of the caspase 3 and the apoptosis rate were significantly increased under B[a]P exposure. In conclusion, these results indicated that ROS was induced by B[a]P, and further triggered the oxidative stress and oxidative damage in hemocytes. B[a]P induced hemocyte apoptosis was mediated by both mitochondrial apoptosis pathway and death receptor apoptosis, and the activation of mitochondrial apoptosis pathway was affected by ROS. In addition, BPDE and MAPKs may play important roles in the B[a]P-mediated apoptosis pathway. This study deepens understanding of the apoptosis pathway and the immunotoxicity mechanism in bivalves hemocytes stimulated by persistent organic pollutants.