Immunotoxicity Mechanism Of Benzo[α]Pyrene On The Haemocyte Of Scallop Chlamys Farreri

Polycyclic aromatic hydrocarbons (PAHs) are one class of toxic environmental pollutants containing two or more than two benzene rings. Although PAHs can originate from natural sources, a major portion of them can be attributed to anthropogenic sources, particularly from fuel combustion, pyrolytic processes, spillage of petroleum products, waste incinerators and domestic heaters. Up to now, there have been more than 400 kinds of carcino-genic PAHs and their ramifications, among which benzo[α]pyrene shows the strongest carcinogenicity. Lately, the pollution condition has been becoming worse because of exploitation of petroleum, development of steamship transportation and outlet of anthropogenic contaminants. According to statistical data, the highest concentration of PAHs in coastal water and sediment can reach up to 551.2 ng·L-1 and 82-4567ng·g-1, respectively, which is at the middle pollution level compared to other countries. The PAHs pollution would make a serious danger to the safety of ocean environment.Scallop (Chlamys farreri) is a main commercial mollusk widely cultured in China. In recent years, the incident of mass mortality of the C. farreri has caused important economic losses. Due to the mussel has filter-feeding habits, low metabolism rate and high accumulation capacity for organic pollutants such as PAHs, some scholars presumed that the large-scale death event of the scallop was related to the pollution in marine environment, however, the scientific support was thin. So this paper studied the effects of B[α]P on several parameters of C. farreri including the haemocyte viability, MAPKs signaling pathways, reactive oxygen species generation, haemocyte damage, haemocyte apoptosis as well as the immune defence index, also this work demonstrated the role of the MAPKs signaling pathway in the process of the cell damage, cell apoptosis and cell immune defence. The following results were obtained: 1 Effects of B[α]P one the haemocyte viability of scallop Chlamys farreriIn the present paper, the suitable detection methods of haemocyte viability and the effects of B[α]P on haemocyte viability of scallop C. farreri were studied. The results showed that when the haemocyte density in 96-well microplate was among 1-32×104 cell per well, the livability of haemocyte was well (as high as 97.42-98.82%), and MTT,XTT and LDH assay were all suitable viability detecting methods of haemocyte. While only the haemocyte density was among 12-32×104 cell per well, the neutral red assay was a suitable detecting method. The cell viability of the primary culture haemocyte in 24h was well using Eagle culture medium. In B[α]P exposure experiment, the neutral red assay showed B[α]P significantly inhibited the haemocyte viability, but the other methods did not detect the inhibition effect, also the results of neutral red assay showed the inhibition extent had a positive correlation with the concentration and exposure time of B[α]P. So it is concluded that the B[α]P had immunotoxicity effects on haemocyte and the neutral red assay could be used as a sensitive index to evaluate the effect of B[α]P on the haemocyte viability of scallop C. farreri.2 The study of the expression of MAPKs signaling pathway and the toxic effects in haemocyte exposed to B[α]PThe effects of B[α]P on MAPKs signaling pathways, ROS generation, lysosomal membrane stability (LMS), actin filament disruption,DNA damage, cell apoptosis of haemocyte as well as immune defence parameters were studied. The results showed that all the parameters were significantly affected by B[α]P, while there was no change in control groups during the experiment. The MAPKs signaling pathways were activated remarkably, the ERK and JNK pathways increased at the beginning of exposure and reached their peaks on 12h in all treatments, following that they all decreased gradually, but were still higher than controls, while the p38 MAPKs was activated earlier than ERK and JNK pathways and showed time- and does- dependent with B[α]P. The ROS generation, the actin filament disruption,DNA damage and cell apoptosis increased during the whole time, and showed positive relationship with the concentration of B[α]P, also the results showed that most of the apoptosis were earlier period apoptosis. While the LMS and the immune defence index were all decreased and showed significant time- and does- dependent with B[α]P. Meanwhile, we observed that the decreased LMS, immune defence index and the increased DNA damage as well as apoptosis had a time-response to the ROS generation, suggesting that the production of ROS during the B[α]P exposure was one of the important causes of cell damage, and the damage as well as apoptosis of haemocyte were important reasons of the decreased immune defence ability. Meanwhile, the author presumed that the activated MAPKs signaling pathway played an important role in the physical response of haemocyte exposed to B[α]P.3 The roles of MAPKs pathwy on the immunotoxicity effects of B[α]PThe roles of MAPKs signaling pathways on the immunotoxicity effects of B[α]P were studied. The results showed that the specific inhibitors of MAPKs could inhibited the activation of the MAPKs pathways, also the MAPKs played an important role in the process of ROS generation, cell damage, cell apoptosis as well as the decreased immune defence ability exposed to B[α]P. The activated ERK and p38 pathways could lead the increase of ROS generation, while the JNK pathway had no effect on it. As to the decreased of LMS, the JNK pathway also had no effect, while the activated ERK decreases the damage and the activated p38 pathway increased the damage of lysosomal membrane. In the process of actin filament disruption, the activated ERK pathway could decrease the damage level, while the activated p38 and JNK pathways played an opposite role compared to the ERK pathway. As to DNA damage, JNK has no effect, while the activated ERK and p38 pathways lead the increased DNA damage. In the process of apoptosis, ERK pathway could decrease the apoptosis, while the p38 and JNK could increase the cell apoptosis. As to the immune defence ability, the activated ERK pathway could enhance the immune ability, while the p38 and JNK pathways had the opposite effect. From the results we could see the roles of MAPKs on the varied responses of haemocyte exposed to B[a]P were different, in some occasion,it played the positive roles, but sometimes it also have the opposite effects. But considering the whole effects of MAPKs on the physical responses, the positive effects were equal to the negative roles in the process of lysosomal membrane damage, while in other responses such as ROS generation, DNA damage, cell apoptosis as well as the decreased immune defence, the positive roles were bigger than the negative roles, suggesting that the activation of MAPKs pathway decreased the immune defence ability of scallop by playing important roles in varied physical responses of haemocyte exposed to B[a]P.