Bioavailability And Toxic Effect Of Benzo[a]pyrene On Portunus Trituberculatus

With the development of maritime petroleum, marine transportation and industry pollution discharge, polycyclic aromatic hydrocarbon(PAHs) pollution became more serious. Benzo[a]pyrene(Ba P) is one of the most carcinogenic PAHs,and widely distributed in the seawater, had been used as a representative study of PAHs. P.trituberculatus is widely cultured in the coast of China as an important economy bivalve. We adopted the seawater-organism systems, study the absorption and release of Ba P in P.trituberculatus using a semi-static two-compartment kinetic model by dynamic detection the Ba P concentration of crab, the change of metabolic detoxification enzyme systems and antioxidant enzyme system(EROD, GST, SOD, MDA),and the gene expression(P4501A1, P-gp, HSP70) on different levels from physiology, biochemistry and molecular biology. then on the validity of these biomarkers from the detection of marine environmental pollution assessment, in order to explore the bioavailability of P.trituberculatus and the toxic effects. The results are as follows:1.The research about the bioavailability of Ba P on Portunus trituberculatusIn this experiment, 3 trestments(0.45 μg·L-1、0.15 μg·L-1、0.05 μg·L-1 of Ba P) were set up to investigate the kinetic parameters of bioconcentrations(including uptake rate constant(K1), elimination rate constant(K2), bioconcentration factor(BCF), and biological half-life(B1/2)), by 15 days of enrichment experiments and the test 15 days of release. The results suggested that Ba P exposure are difference on Ba P contents in P.trituberculatus, and the treatment of 0.45 μg·L-1 had obvious difference compared with the control group(P<0.05), while the Ba P content in crab was no significant difference by compared the group of 0.05μg·L-1 and the control group(P>0.05). During the enrichment-elimination trials, the result showing the law of bell curve among all treatments. Besides, the modeling results showed that K1 ranged from 14.67 to 26.08, K2 ranged from 0.09 to 0.14, maximal content(CAmax) ranged from 9.31 to 73.35, BCF ranged from 163.00 to 196.29, and B1/2 ranged from 4.95 to 7.70 d. We found that the Ba P concentration data was confirmed to the two-compartment model, and that good accordance in the result from the calculate and observed the Ba P of concentrations using the goodness-of-fit test. The result also calculate that K1 and BCF of P.trituberculatus generally decreased with the increase of Ba P reveal consistence in the seawater, K2 had no significant relationship with the Ba P concentrations in the seawater, and that maximal content in the organism at the theoretic increased and was basically proportional to increasing Ba P concentration in the water. The semi-static two-compartment kinetic model could well fit the bioconcentration of Ba P in P.trituberculatus about the experimental result showed. The enrichment experiments show P.trituberculatus has a strong enrichment capability for Ba P, the absorption rate constant and elimination rate constant were reducing with the seawater concentration increases. The maximal content were increases with the growing of Ba P concentration. During the release experiments, with the increase of time the elimination rate constant became slow and the Ba P in organism gradually reduce close to the control group. Whatever the content of Ba P in the treatment of 0.45 μg·L-1 is still high. Further the biological half-life exposure with increasing Ba P concentration increases.2. The influence mechanim of detoxification and antioxidant enzyme on Portunus trituberculatus exposed to different Benzo[a]pyrene concentrationsAfter 15 days of enrichment experiments in different Benzo[a]pyrene concentrations(0.45 μg·L-1,0.15 μg·L-1,0.05 μg·L-1 of Ba P), P.trituberculatus transferred to clean seawater conducted 15 days of the release. in the first 0 days, 1 day, 3 days, 5 days, 10 days, 15 days the P.trituberculatus’ s gill, liver was detected for various metabolic detoxification enzymes and antioxidant enzyme s(7-ethoxyresorufin-O-deethylase(EROD), glutathine S-transferease(GST), superoxide dismutase(SOD), lipid peroxidation(LPO)) activity. Result showed that the time-response trend on of induction of hepatic EROD and GST of P.trituberculatus was bell-shaped with a plateau at Ba P exposure, the hepatic of SOD and MDA were increase with the time. The enzymes in gill of induction have a similar law, but not identical. The EROD, GST and SOD activity were enhanced at maximum at 5days, 10 days and 15 days, and then inhibited gradually in the gill after a 3 d exposure. At the same time, the induction of gill MDA was in increase with the time. Furthermore,the EROD, GST, SOD and MDA activity showed a significant induction(P<0.05) with the Ba P increase in P.trituberculatus of gill and liver,and have an obvious dose-effect.In the elimination period, various detoxification and antioxidant enzymes were rapidly reduced in the different concentrations of Ba P, and the enzymes activity in P.trituberculatus was recovery with 0.05 μg·L-1 of Ba P. Moreover, the EROD, GST and MDA activity were recovery the level of the control group in the gill with 0.15 μg·L-1 and 0.45 μg·L-1 of Ba P, and the SOD activity showed a significant induction(P<0.05)compared with the control group. The EROD and MDA activity were recovery the level of the control group in the liver with 0.15 μg·L-1 and 0.45 μg·L-1 of Ba P, and the SOD and GST activity showed a significant induction(P<0.05) compared with the control group.3.Effect of Benzo[a]pyrene on the mechanim of detoxification and antioxidant genes expression in Portunus trituberculatusThe P.trituberculatus was exposed to the water of Ba P at different concentrations(0.45 μg·L-1,0.15 μg·L-1,0.05 μg·L-1).After a period of 15 d, the crab was removed to the natural sea water. During the experiment period, take samples to measure the concentrations of Ba P in P.trituberculatus.Result showed the gene expression patterns of P4501A1, P-gp and HSP70 of P.trituberculatus were both measured using realtime fluorescence quantitative PCR during crab exposed to environmentally relevant concentration of Ba P(0.05 μg·L-1,0.15 μg·L-1,0.45 μg·L-1, respectively) and the release of the test.The results showed that P451A1, P-gp and HSP70 m RNA could be induced, besides, the content of P4501A1 m RNA induced is more significantly(P<0.05). The content of three genes m RNA were decrease markedly after the release of the test and the P4501 A m RNA has more obvious changes. In a word, The gene expression of P4501A1, P-gp and HSP70 were increase at the beginning of Ba P exposure, and then decreased after the release of the test. The above data suggest that Ba P could induced high concentration of P451A1 m RNA in the liver of crab to monitor indicates contamination of organic pollutants in the water of PAHs.