Biological Monitoring Technology Research Of Offshore PAHs In The Clam Ruditapes Philippinarum

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants (POPs), containing two or more benzene hydrocarbons. PAHs are known for their carcinogenic, teratogenic and mutagenic properties. The cumulative effects of PAHs in the marine organisms will not only endanger themselves, may eventually threaten human health through the food chain. In 2011, the European Food Safety Authority (EFSA) has introduced a system of four specific PAHs, namely benzo[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF) and chrysene (Chr), assessing that the sum of the four PAH compounds would be the most suitable indicator for PAHs in food. The clam Ruditapes philippinarum is an important commercial species in China. Our current study used the Illumina’s sequencing and gene cloning technology to determine the molecular mechanism of metabolic detoxification in the clam R. philippinarum under PAHs exposure. The main objective of this study was to investigate the accumulation and elimination rules of PAHs, to select useful biochemical biomarkers for PAHs pollution monitoring, and to demonstrat the effectiveness of biomarkers through field sampling experiments in the Jiaozhou Bay and Laizhou Bay of China. The resulting data may provide technical support for marine environmental pollution monitoring and assessment of aquatic products safety.1 Study on molecular mechanism of clam R. philippinarum exposed to PAHsIn this study, digital gene expression (DGE) was performed to investigate the digestive gland transcriptome profile of the clam Ruditapes philippinarum exposed to BaP. A total of 10,508,312 and 11,414,297 clean reads were generated respectively, from control and BaP exposure DGE libraries. One hundred and forty-five differentially expressed genes were detected after comparing two libraries with 58 up-regulated, and 87 down-regulated genes. GO annotation and KEGG pathway analyses were performed on all genes to understand their biological functions and processes. The results showed that numerous enriched differentially expressed genes are related to growth and development, antioxidant metabolism, apoptosis and detoxification metabolism. Quantitative real-time PCR was performed to verify the expressed genes of DGE.The present research has obtained full-length cDNAs of genes HSP70 and HSP 90 from the clam R. philippinarum. The full-length RpHSP70 cDNA was 2336 bp containing a 5’ untranslated region (UTR) of 51 bp, a 3’UTR of 335 bp and an open reading frame (ORF) of 1950 bp encoding 650 amino acid residues. The full-length RpHSP90 cDNA was 2839 bp containing a 107-bp 5’UTR, a 554-bp 3’UTR and a 2178-bp ORF encoding 726 amino acid residues. The deduced amino acid sequences of RpHSP70 and RpHSP90 shared the highest identity with the sequences of Paphia undulata, and the phylogenetic trees showed that the evolutions of RpHSP70 and RpHSP90 were almost in accord with the evolution of species. The RpHSP70 and RpHSP90 mRNA expressions were detected in all tested tissues in the adult clams (digestive gland, gill, adductor muscle and mantle) and the highest mRNA expression level was observed in the digestive gland compared to other tissues. Quantitative real-time RT-PCR analysis revealed that mRNA expression levels of the clam RpHSP70, RpHSP90 in the digestive gland of R. philippinarum.2 Study on the PAHs Biomarker screening technology basing on the clam R. ph ilippinarumIn the present study, we use the clam R. philippinarum as a model organism, which were exposed to PAHs (mixing ratio:BaP:BbF:Chr:BaA=1:1:1:1, concentration gradient:0,0.05, 0.5,5 μg/L) for 21days (sampled at 0,1,3,6,10,15,21d) and depurated for 15days (sampled at 22,24,27,31,36d). The clam tissues, including the digestive gland, gill, soft tissue and adductor muscle, were sampled. The PAHs concentration in the four tissues were analysed using liquid-liquid extraction and HPLC. All the results indicated that a clear time effect with exposure concentration was established for PAHs. During the PAHs exposure time, the concentrations of the four PAHs in the same tissue followed the rules:Chr> BaA> BaP> BbF. The total concentrations of PAHs in the four tissues followed the rules:digestive gland> gill> soft tissue> adductor muscle. When the depuration experiment initiated, the accumulation of PAHs dropped significantly. During the whole experiment, the control group had no significant change.The purpose of this study was to investigate the impact of PAHs on metabolic detoxification system of the clam R. philippinarum. (i) detoxification enzyme activities of phase I (AHH, DD, EH) and phase II (GST, UGT, SULT); (ii) Antioxidant defense system (T-AOC, SOD, GSH/GSSG); (iii) levels of oxidative stress parameters (F value, MDA, PC); (iv) expression levels of genes (AhR, HSP90, ARNT, EH, DD, GST, SULT, PgP, SOD) were determined, and results showed that all the parameters were induced in a dose-dependent marine.3 Study on the offshore PAHs biological monitoring technology of of Jiaozhou bay and Laizhou bay basing on the clam R. philippinarum.The results in offshore of Jiaozhou Bay (Red Island and Ying Hai) and Laizhou Bay (Guangli harbor and Xiaying harbor) in the the four seasons (March, June, September, December) showed that PAHs pollution in Yinghai was the most serious in Jiaozhou Bay. The results in Laizhou Bay showed that PAHs pollution in Guangligang was the most serious. PAHs concentrations in June and September were the highest, but season hardly affected PAHs accumulation and biomarkers in R. philippinarum. According to the correlation analysis, AHH activity, MDA content, F value and mRNA expression of GST gene had good correlation with PAHs accumulation in R. philippinarum. As a result, they can be used as useful biomarkers in marine PAHs monitoring.