Study On Mechanism For Reproductive Endocrine Disrupting Effects Of Benzo[α]pyrene On Scallop Chlamys Farreri

Polycyclic aromatic hydrocarbons (PAHs) belong to Persistent Toxic Substances(PTS), including various substances that have carcinogenic teratogenic and mutageniceffects. Among PAHs, benzo[α]pyrene (B[α]P) is the most carcinogenic one and manystudies also show that B[α]P has endocrine disrupting effects. At present studies onphenomenon and mechanism of endocrine disruption are concentrate on mammals andfish but deficient in invertebrates both at home and abroad. In this study, B[α]P was used asthe representative of PAHs to investigate its influences on steroid levels in gonad andhaemolymph, steroidogenic enzymes genes and related nuclear receptors mediatedgenes expression in ovary, DNA strand breakage, peroxide damage and histological ofChlamys farreri gonad and was discussed. The following results were obtained:1Effects of B[α]P on sex steroids in C. farreri gonad and haemolymphIn this study, scallops were exposed to different concentrations of B[α]P (0.025μg/L,0.5μg/L,10μg/L) for10days, and sampled on day3and day10. Progesterone,testosterone and17β-estradiol levels in gonad and haemolymph were identified andquantified by electrochemiluminescence immunoassay (ECLIA) method. Theseobservations suggest that exposure to B[α]P disturbs progesterone levels in gonad, andevery dose B[α]P exposure can exerts great suppression on testosterone,17β-estradiolproduction at3d and10d. At10d17β-estradiol and testosterone levels in haemolymphare also affected by B[α]P exposure but they are relatively less susceptible duringexperimental period. The effect of B[α]P on males is higher than its influence onfemales, which shows that sex steroids in males have more significantly or higherchanges than in females when exposing to same dose B[α]P. Present study shows thatB[α]P has endocrine disrupting effects via disturbing progesterone and inhibitingtestosterone and17β-estradiol levels. In addition, B[α]P has more severe effects onmales than females.2Effects of B[α]P on reproduction relevant genes expression in C. farreri ovaryPresent study obtains complete C. farreri CYP3A cDNA sequence through the technology of RT-PCR and RACE. The full-length Cf CYP3A cDNA is2110bp,containing an open reading frame (ORF) of1578bp which encoded a polypeptide of525amino acids. Amino acid analysis shows the identity of C. farreri CYP3A comparedwith Saccoglossus kowalevskii CYP3A13-like, CYP3A90-like and Strongylocentrotuspurpuratus CYP3A8-like is42%,40%and40%respectively, so it is classified intoCYP3A family. Members of the vertebrate CYP3A subfamily are involved in themetabolism of steroids and a wide range of xenobiotics. In this study, scallops wereexposed to different concentrations of B[α]P (0.025μg/L,0.5μg/L,10μg/L) for10days,and sampled on day3and day10. Reproduction relevant genes expression were testedby real time quantitative PCR. Results showed that long time B[α]P exposure decreasessteroidogenic enzymes,3β-HSD, CYP17and17β-HSD, genes expression in adose-dependent manner, which is consistent with previous research. Therefore it issuggested that they could be potential targets of B[α]P in the C. farreri ovary thatdisrupt steroidogenic machinery. In addition, it shows that0.025μg/L B[α]Pup-regulates AhR, ARNT, CYP1A1, CYP3A, ER and Vg gene expression, whichsuggests that0.025μg/L B[α]P can not only activate AhR-mediated transcription butalso induce estrogenicity, but10μg/L B[α]P inhibits these genes expression. It isspeculated that long time high dose B[α]P exposure induce toxicology which is toosevere for the organism to deal with, and then the AhR signaling could not be activatedto mediate genes responsible for detoxification up-regulating. This kind of pollutionbeyond organism regulating ability could lead to collapse of detoxification and defencesystems and severe damage finally. ER-Vg genes expression up-regulating induced byB[α]P may be mediated via aryl hydrocarbon receptor (AhR) inducedB[α]P-metabolising enzymes CYP1A1and1B1transcriptional activation. But10μg/LB[α]P could surpress ER-Vg genes expression ER-Vg genes expression, it may bebecause that as the B[α]P exposure levels increasing, B[α]P metabolism and toxicityincreased so as to modify ER-signaling and then instead ER and Vg mRNA expressiondecreased. And that according to experimental results as the B[α]P exposure levelsincreasing the T and E2synthesis are severely inhibited, abnormal low levels of sexhormones may also affect ER signaling pathway in ovaryof C. farreriand finally resultin low expression of ER and VTG mRNA.3. Damage Effects of B[a]P in gonad of C. farreriIn this study, scallops were exposed to different concentrations of B[α]P (0.025μg/L, 0.5μg/L,10μg/L) for10days, and sampled on day0,1,3,6and10. The levels of DNAstrand breaks in the female ovary, oxidative carbonyl protein and LPO levels in bothovary and spermary were monitored.0.5μg/L and10μg/L B[α]P exposure result inhigher DNA strand breakage in a dose-dependent manner and cannot be repaired duringexperimental period. Every B[α]P treatment induces oxidative carbonyl protein andLPO levels increase in a dose-dependent manner but decrease over exposure time.Oxidative carbonyl protein and LPO levels in0.025μg/L and0.5μg/L B[α]P treatmentscan decrease to control levels but except for10μg/L B[α]P treatment. Among theseinvestigations it shows that male spermary is more sensitive than female ovary becausesame B[α]P treatment can induce more significantly oxidant stress or more difficult tobe repaired in spermary.4Effects of B[α]P on microstructure of C. farreri gonadIn this study, scallops were exposed to different concentrations of B[α]P (0.025μg/L,0.5μg/L,10μg/L) for10days, and sampled on day10. Histological analysis of theovaries indicated that after10days0.5and10μg/L B[a]P leaded to more degeneratingoocytes and immature oocytes in the outer layer of the ovaries. Furthermore exposure to10μg/L B[a]P the connective tissue of the ovary envelops became damaged, and theinterspace among follicles was biggest compared to all other B[a]P exposuretreatments.. Follicles in spermary also have bigger interspace and not full with spermslike control. It demonstrats that the development of ovary was inhibited significantlyand damaged by B[α]P toxicology.