Glutathione transferase pi class 2 (GSTp2) protects against the cardiac deformities caused by exposure to PAHs but not PCB-126 in zebrafish embryos

Glutathione transferases (GSTs) are phase II enzymes that detoxify a wide range of toxicants and reactive intermediates. One such class of toxicants is the ubiquitous polycyclic aromatic hydrocarbons (PAHs). Certain PAHs are known to cause developmental cardiac toxicity in fish. Herein, we explored the role of GST pi class 2 (GSTp2) in PAH- and PCB-induced cardiac toxicity in zebrafish (Danio rerio) embryos. We measured expression of GSTp2 in embryos exposed to individual and co-exposures of the PAHs benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), and fluoranthene (FL) as well as 3,3',4,4',5-pentachlorobiphenyl (PCB-126). GSTp2 mRNA expression was induced by exposure to BkF, BaP, PCB-126, and BaP+FL and BkF+FL co-exposure. A splice junction morpholino was then used to knockdown GSTp2 in developing zebrafish. GSTp2 knockdown exacerbated the toxicity caused by co-exposures to BkF+FL and BaP+FL. However, GSTp2 knockdown did not affect PCB-126 toxicity. These results further suggest that pi class GSTs serve a protective function against the synergistic toxicity caused by PAHs in developing zebrafish.     

Mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) in the gill and hepatopancreas of rpsL transgenic zebrafish

We examined the in vivo mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) by using transgenic (Tg) zebrafish carrying the mutational target gene rpsL. PBTA-6 is one of the PBTA-type compounds that were recently identified in highly mutagenic river water in Japan. BaP is a well-known contaminant that is frequently found in polluted water. Both compounds are potent mutagens, as determined by using the Ames test employing S9 mix and Salmonella. Adult rpsL Tg zebrafish were exposed to 0, 7, or 10 mg/L PBTA-6 or 0, 1.5, or 3 mg/L BaP for 96 h in a water bath and the mutations in their gills and hepatopancreata were measured 2-4 weeks later. At 3 weeks after exposure, 3 mg/L BaP significantly increased the rpsL mutant frequency (MF) in the gill and hepatopancreas by 5- and 2.3-fold, respectively, as compared to control fish. Sequence analysis showed that BaP mainly induced G:C to T:A and G:C to C:G transversions, which is consistent with the known mutagenic effects of BaP. In contrast, despite its extremely high mutagenic potency in Salmonella strains, PBTA-6 did not significantly increase the MF in the zebrafish gill or hepatopancreas. Although PBTA-6 is 300 times more mutagenic than BaP in the Ames test [T. Watanabe, H. Nukaya, Y. Terao, Y. Takahashi, A. Tada, T. Takamura, H. Sawanishi, T. Ohe, T. Hirayama, T. Sugimura, K. Wakabayashi, Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan, Mutat. Res. 498 (2001) 107-115], calculation of the mutagenicity per mole of compound indicated that PBTA-6 was 33- and <3.7-fold less mutagenic in the zebrafish gill and hepatopancreas, respectively, than BaP.     

Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.     

Benzo(a)pyrene inhibits endometrial cell apoptosis in early pregnant mice via the WNT5A pathway

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg⁻¹·day ⁻¹ BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function.     

Transgenic expression of tilapia hepcidin 1-5 and shrimp chelonianin in zebrafish and their resistance to bacterial pathogens

Recently, tilapia hepcidin (TH)1-5 was characterized, and its antimicrobial functions against several pathogens were reported. The antimicrobial functions of another shrimp antimicrobial peptide (AMP), chelonianin, were also characterized using a recombinant chelonianin protein (rcf) that was expressed by a stably transfected Chinese hamster ovary (CHO) cell line against pathogen infections in fish. The function of the overexpression of both AMPs in zebrafish muscles was not examined in previous studies. Herein, we investigated the antimicrobial functions of TH1-5 and chelonianin against Vibrio vulnificus (204) and Streptococcus agalactiae (SA48) in transgenic TH1-5 zebrafish and transgenic chelonianin zebrafish. The presence of TH1-5 and chelonianin enhanced the inhibitory ability in transgenic AMP zebrafish against the two different bacterial infections. The bacterial number of either V. vulnificus (204) or S. agalactiae (SA48) had decreased at 96?h after injection into transgenic AMP zebrafish muscle compared to non-transgenic zebrafish muscle. Additionally, immune-related gene expressions analyzed by real-time PCR studies showed the modulation of several genes including interleukin (IL)-10, IL-22, IL-26, MyD88, Toll-like receptor (TLR)-1, TLR-3, TLR-4, nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, and lysozyme, and significant differences were found between transgenic AMP zebrafish and wild-type zebrafish injected with PBS at 1-24?h. These results suggest that several immune-related gene expressions were induced in transgenic TH1-5 and chelonianin zebrafish which effectively inhibited bacterial growth. The survival rate dropped to 86.6% in transgenic chelonianin zebrafish after 28 days of infection compared of the 50% survival rate in transgenic TH1-5 zebrafish after 28 days of infection. Overall, these results indicate that TH1-5 and chelonianin possess the potential to be novel candidate genes for aquaculture applications to treat fish diseases.     

Osmolarity and composition of cell culture media affect further development and survival in zebrafish embryos

With the aim of carrying out chimaerism and somatic cell-midblastula transition (MBT) embryos co-culture experiments in freshwater fish species, we evaluated the effect of osmolarity and composition of two media commonly used in cell fish culture on MBT zebrafish embryos and their further development and survival. To this end, wild zebrafish dechorionated embryos in midblastula stage were cultured for 6 days (Experiment 1: 189 embryos) or 1 h (Experiment 2: 150 embryos) in three different media: Hanks' 10% (H-10), 35 mOsm; Hanks' 100% (CH), 315 mOsm; and L-15 with serum (L-15: 315 mOsm). High osmolarity affected the survival rate (6 days: L-15: 45.1% v. CH: 72.34% v. H-10: 100%, P < 0.05; after 6 days: 0% both in L-15 and CH) and slowed their developmental timing. Embryos showed tail deformation (curly) as well as body paralysis at 48 h when they showed tail movements at 28 h. Differences in tail deformation were observed between high-osmolarity groups (CH: 85.10% v. L-15: 98.04%; P < 0.05). In Experiment 2, no effects on survival rate were observed. Teratogenic effects were only observed in L-15 (L-15: 12.98% v. CH: 0%; P< 0.05). Loss of motility was not detected in any group at 48 h. Optimum osmolar condition for cultured cells and also embryonic cells is around 315 mOsm and so, during chimaerism experiments (usually practised at MBT stage), present results indicate that midblastula embryos can acceptably bear the effects caused by 315 mOsm (CH) for 1 h, even though this involves a certain delay in developmental timing.     

Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle

In the present study, new applications of the transgenic technology in developing novel varieties of ornamental fish and bioreactor fish were explored in a model fish, the zebrafish (Danio rerio). Three "living color" fluorescent proteins, green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP or dsRed), were expressed under a strong muscle-specific mylz2 promoter in stable lines of transgenic zebrafish. These transgenic zebrafish display vivid fluorescent colors (green, red, yellow, or orange) visible to unaided eyes under both daylight and ultraviolet light in the dark. The level of foreign protein expression is estimated between 3% and 17% of total muscle proteins, equivalent to 4.8-27.2mg/g wet muscle tissue. Thus, the fish muscle may be explored as another useful bioreactor system for production of recombinant proteins. In spite of the high level of foreign protein expression, the expression of endogenous mylz2 mRNAs was not negatively affected. Furthermore, compared to the wild-type fish, these fluorescent transgenic fish have no advantage in survival and reproduction.     

三氯乙烯对斑马鱼胚胎心脏发育的毒性作用及机制*

目的:研究三氯乙烯(TCE)对斑马鱼胚胎心脏发育的毒性作用及其机制,为寻找干预靶点提供实验依据。方法:斑马鱼胚胎来自于国家斑马鱼资源中心,分为DMSO组(对照组)、DMSO+CHIR组、DMSO+XAV组、TCE处理组、TCE+CHIR组和TCE+XAV组(TCE设置为1、10、100 ppb三个不同的浓度;DMSO:二甲基亚砜;CHIR:CHIR-99021,Wnt信号通路激活剂;XAV:XAV-939,Wnt信号通路抑制剂),每组60条。斑马鱼胚胎饲养于系统养殖水中,恒温28℃,每隔24 h更换养殖水,并分别加入相应药物。连续培养72 h,收集斑马鱼胚胎的心脏组织,提取RNA进行转录组芯片分析,并以荧光定量PCR验证Wnt信号通路相关基因的表达。结果:与对照组相比,三氯乙烯暴露导致斑马鱼心脏畸形显著增加,以心房心室比例异常、环化不全以及心包水肿等为主要表型。芯片分析结果显示,TCE处理组Wnt信号通路相关基因(Axin2、Sox9b、Nkx2.5)表达受到显著影响。qPCR结果进一步验证,TCE处理组与DMSO对照组相比,Wnt通路靶基因Axin2、Sox9b及Nkx2.5的mRNA水平显著下调(P＜0.05),提示Wnt信号通路被抑制。Wnt激活剂CHIR降低TCE导致的斑马鱼胚胎心脏发育异常,而添加Wnt通路抑制剂XAV后,斑马鱼胚胎心脏畸形率显著增加(P＜0.05)。结论:三氯乙烯暴露导致斑马鱼胚胎心脏畸形,Wnt信号通路参与三氯乙烯的心脏发育毒性。     

Transgenic Zebrafish Expressing Chicken Lysozyme Show Resistance against Bacterial Diseases

We established a transgenic zebrafish strain expressing chicken lysozyme gene under the control of the Japanese flounder keratin gene promoter, and investigated its resistance to a pathogenic bacterial infection. To generate the lysozyme transgenic construct, Japanese flounder keratin promoter was linked to both the hen egg white (HEW) lyoszyme gene and green fluorescence protein (GFP) gene used as a selection marker for the transgenic strains, in a recombinant plasmid. The recombinant plasmid was microinjected into fertilized zebrafish eggs. In F2 transgenic zebrafish, GFP expression was strong in the epithelial tissues, liver and gill from the embryonic stage to the adult stage. The expressions of HEW lysozyme and GFP mRNA were confirmed in the liver and skin by RT-PCR. Western blot analysis showed that both HEW lysozyme and GFP were present in protein extracts from the liver of transgenic zebrafish, but not in protein extracts from the muscle. The lytic activity of protein extracts from the liver (assessed by a lysoplate assay using Micrococcus lysodeikticus as a substrate) was 1.75 times higher in F2 transgenic zebrafish than in the wild type. In a challenge experiment, 65% of the F2 transgenic fish survived an infection of Flavobacterium columnare and 60% survived an infection of Edwardsiella tarda, whereas 100% of the control fish were killed by both pathogens. However, the survival rates of the transgenic fish were not significantly higher when higher concentrations of bacteria were used.     

Interaction of benzo[a]pyrene, 2,3,3',4,4',5 hexachlorobiphenyl (PCB 156) and cadmium on biomarker responses in flounder (Platichthys flesus L.)

Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg^-1), 2,3,3',4,4'5 hexachlorobiphenyl (PCB-156, 2.5 mg kg^-1), and cadmium (cadmium, 1 mg kg^-1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB treated flounder after 2 and 8 days, respectively. The strongest induction (44 fold) was caused by BaP. No further induction was observed after additional treatment with PCB 156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre treated with cadmium, whereas induction by PCB 156 appeared to be unaffected by pre treatment with cadmium. Flounder treated with cadmium only had significantly elevated hepatic levels of metallothionein (MT) after 15 days. Pre treatment with BaP and PCB prior to cadmium inhibited the MT induction (30-50%) compared with cadmium alone. Furthermore, significantly higher glutathione S transferase activities were observed in flounder administered cadmium alone, and in flounder treated with BaP or PCB 156 prior to cadmium. GST selenium independent peroxidase activities appeared to be unaffected by any of the treatments in the present study. The results indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone, and that the sensitivity of both CYP1A and MT are influenced by pollutants other than their primary inducers.     

Regulator of complement activation (RCA) group 2 gene cluster in zebrafish: identification, expression, and evolution

The activation of the complement system is tightly regulated by a group of plasma and cell membrane-associated proteins for host cell protection. In humans, these regulatory protein genes are clustered in a region named the regulator of complement activation (RCA) gene locus and can be categorized into two groups. The group 1 gene cluster has been reported in zebrafish, but information regarding the RCA locus remains scarce in fish. Here we identified two closely linked RCA group 2 genes in zebrafish, ZRC1 and ZRC2, which had all the features characteristic of known RCA group 2 genes. Both ZRC1 and ZRC2 were closely linked to the PFKFB1 gene and located 17 Mkb downstream of the PFKFB2 gene; in contrast, RCA group 2 genes are closely linked to PFKFB2 in frogs, chickens, and humans. However, both the direction of the RCA group 2 genes relative to PFKFB2 and the order of the RCA group 2 gene-encoded proteins in zebrafish were comparable to those in frogs, chickens, and humans. ZRC1 and ZRC2 shared 71.1% identity to each other, implicating that they might have originated by gene duplication after the split of the fish/mammalian common ancestor. Moreover, ZRC1 and ZRC2 encoded a membrane-associated protein and a soluble protein, respectively, and displayed different expression patterns, suggesting that functional divergence has already occurred. This is the first report showing the presence of the RCA group 2 cluster as well as the membrane-associated complement regulatory protein in zebrafish, providing a better understanding of the origin and evolution of RCA proteins.     

Southern flounder hepatic and intestinal metabolism and DNA binding of benzo[a]pyrene (BaP) metabolites following dietary administration of low doses of BaP, BaP-7,8-dihydrodiol or a BaP metabolite mixture

Certain finfish species living in chemically polluted environments exhibit a high incidence of gastrointestinal tract tumors. Carnivorous fish in such environments are likely to consume invertebrates which contain chemical procarcinogens and the invertebrate biotransformation products of these compounds. The retention in tissues, extent of DNA adduct formation in liver and intestine, and metabolite composition of bile was investigated in southern flounder following gavage with pure [3H]- or [14C]benzo[a]pyrene (BaP), pure [14C]benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8D), or hepatopancreas from spiny lobsters previously dosed with [3H]- or [14C]BaP (Metab.HP). Metab.HP contained mainly polar conjugates of BaP diols, triols and tetraols. BaP-7,8D was retained in fish tissues and bile at 24 h to a greater extent (33.6% of the dose), than either BaP (19.00%) or Metab.HP (6.6%). Hepatic and intestinal DNA isolated from all dosed fish contained covalently bound radioactivity, but exposure to BaP-7,8D or BaP resulted in significantly higher binding in both tissues than exposure to Metab.HP. Hepatic DNA from BaP and BaP-7,8D-dosed flounder contained 0.24 +/- 0.07 and 0.33 +/- 0.06 pmol BaP equivalents/mg DNA respectively (mean +/- S.E.), while hepatic DNA isolated from Metab.HP-dosed flounder contained 0.006 +/- 0.002 pmol BaP equivalents/mg DNA. Binding of radioactivity to intestinal DNA was significantly higher than to hepatic DNA for flounder dosed with Metab.HP (0.026 +/- 0.003) or with BaP (0.76 +/- 0.27) but not for flounder dosed with BaP-7,8D (0.44 +/- 0.09). These studies show that dietary BaP, and metabolites likely to be present in invertebrates, can be absorbed by the southern flounder and form DNA adducts in target organs.     

Comparison of dietary and waterborne exposure to benzo a pyrene: bioavailability,tissue disposition and CYP1A1 induction in rainbow trout Oncorhynchus mykiss

Absorption and tissue distribution of benzo\[ a ]pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.     

Critical developmental stages for the efficiency of somatic cell nuclear transfer in zebrafish

Somatic cell nuclear transfer (SCNT) has been performed extensively in fish since the 1960s with a generally low efficiency of approximately 1%. Little is known about somatic nuclear reprogramming in fish. Here, we utilized the zebrafish as a model to study reprogramming events of nuclei from tail, liver and kidney cells by SCNT. We produced a total of 4,796 reconstituted embryos and obtained a high survival rate of 58.9-67.4% initially at the 8-cell stage. The survival rate exhibited two steps of dramatic decrease, leading to 8.7-13.9% at the dome stage and to 1.5-2.96% by the shield stage. Concurrently, we observed that SCNT embryos displayed apparently delayed development also at the two stages, namely the dome stage (1:30 ± 0:40) and the shield stage (2:50 ± 0:50), indicating that the dome and shield stage are critical for the SCNT efficiency. Interestingly, we also revealed that an apparent alteration in klf4 and mycb expression occurred at the dome stage in SCNT embryos from all the three donor cell sources. Taken together, these results suggest that the dome stage is critical for the SCNT efficiency, and that alternated gene expression appears to be common to SCNT embryos independently of the donor cell types, suggesting that balanced mycb and klf4 expression at this stage is important for proper reprogramming of somatic nuclei in zebrafish SCNT embryos. Although the significant alteration in klf4 and mycb expression was not identified at the shield stage between ZD and SCNT embryos, the importance of reprogramming processes at the shield stage should not be underestimated in zebrafish SCNT embryos.     

Evolutionary analysis of putative olfactory receptor genes of medaka fish, Oryzias latipes

To obtain an understanding of the origin, diversification and genomic organization of vertebrate olfactory receptor genes, we have newly cloned and characterized putative olfactory receptor genes, mfOR1, mfOR2, mfOR3 and mfOR4 from the genomic DNA of medaka fish (Oryzias latipes). The four sequences contained features commonly seen in known olfactory receptor genes and were phylogenetically most closely related to those of catfish and zebrafish.Among them, mfOR1 and mfOR2 showed the highest amino acid (aa) similarity (93%) and defined a novel olfactory receptor gene family that is most divergent among all other vertebrate olfactory receptor genes. Southern hybridization analyses suggested that mfOR1 and mfOR2 are tightly linked to each other (within 24kb), although suitable marker genes were not available to locate their linkage group. Unlike observation in catfish olfactory receptor sequences, nucleotide (nt) substitutions between the two sequences did not show any evidence of positive natural selection. mfOR3 and mfOR4, however, showed a much lower aa similarity (26%) and were both mapped to a region in the medaka linkage group XX.After including these medaka fish sequences, olfactory receptors of terrestrial and aquatic animals formed significantly different clusters in the phylogenetic tree. Although the member genes of each olfactory receptor gene subfamily are less in fish than that in mammals, fish seem to have maintained more diverse olfactory receptor gene families. Our finding of a novel olfactory receptor gene family in medaka fish may provide a step towards understanding the emergence of the olfactory receptor gene in vertebrates.     

Formation and persistence of benzo[a]pyrene-diolepoxide-DNA adducts in liver of English sole (Parophrys vetulus)

The formation of DNA adducts from the carcinogenic environmental pollutant benzo[a]pyrene (BaP) was investigated in liver of English sole (Parophrys vetulus), a fish species that exhibits a high prevalence of liver neoplasms in several polycyclic aromatic hydrocarbon (PAH)-contaminated areas of Puget Sound, WA. Analysis by the 32P-postlabeling assay of hepatic DNA digests from English sole exposed parenterally to BaP showed the presence of BaP-diol epoxide (BaPDE)-DNA adducts. When English sole were injected with 2-15 mg BaP/kg body wt., one major adduct was detected and was identified as the anti-BaPDE-DNA adduct. Moreover, in English sole sampled at 1, 28 and 60 days post-exposure to 15 mg BaP/kg body wt., there was no significant change in the level of the anti-BaPDE-DNA adduct. The autoradiographs of 32P-labeled hepatic DNA digests from fish exposed to 100 mg BaP/kg body wt. showed an elongated spot suggesting the presence of more than one adduct. Chromatography on large polyethyleneimine sheets (20 x 20 cm) showed 2 spots with the same chromatographic characteristics as those of syn- and anti-BaPDE-deoxyguanosine adduct standards. Mild acid hydrolysis of hepatic DNA of English sole, exposed to 100 mg BaP/kg body wt., also revealed the presence of tetrols derived from both anti- and syn-BaPDE, thus confirming the presence of syn- and anti-BaPDE. In fish exposed to 2-100 mg BaP/kg body wt., a linear (0.996) dose response for anti-BaPDE-DNA adduct formation was observed. The results from this study offer the first direct evidence for the formation of the suspected ultimate carcinogen, BaPDE, in liver of English sole exposed to BaP in vivo and thus further support the hypothesis that exposure to PAHs is an important factor in the etiology of hepatic neoplasms in English sole from contaminated sites.     

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.     

Uncoupling protein 2 from carp and zebrafish, ectothermic vertebrates.

Uncoupling protein 1 (UCP1) is of demonstrated importance in mammalian thermogenesis, and early hypotheses regarding the functions of the newly discovered UCP homologues, UCP2, UCP3 and others, have focused largely on their potential roles in thermogenesis. Here we report the amino acid sequences of two new UCPs from ectothermic vertebrates. UCPs from two fish species, the zebrafish (Danio rerio) and carp (Cyprinus carpio), were identified in expressed sequence tag databases at the European Molecular Biology Laboratory. cDNAs from a C. carpio 'peritoneal exudate cell' cDNA library and from a D. rerio 'day 0 fin regeneration' cDNA library were obtained and fully sequenced. Each cDNA encodes a 310 amino acid protein with an average 82% sequence identity to mammalian UCP2s. The fish UCP2s are about 70% identical to mammalian UCP3s, and 60% identical to mammalian UCP1s. Carp and zebrafish are ectotherms--they do not raise their body temperatures above ambient by producing excess heat. The presence of UCP2 in these fish thus suggests the protein may have function(s) not related to thermogenesis.     

苯并（a）芘对大弹涂鱼肝细胞超微结构的影响

在实验生态条件下,研究不同浓度苯并(a)芘(BaP)暴露下大弹涂鱼肝脏细胞超微结构的变化。结果表明,暴露于低浓度(0．5mg·L^-1)BaP 7d,大弹涂鱼肝脏细胞内的细胞器受到不同程度的损伤,其中线粒体和内质网是受BaP暴露影响最明显的细胞器,细胞核也受到不同程度的影响,细胞质中脂滴也增加;而暴露于高浓度(5mg·L^-1)BaP 2h,不仅是线粒体和内质网,几乎所有细胞器都受到严重影响,细胞器严重退化,细胞结构遭到严重破坏。研究结果证实,BaP可对大弹涂鱼肝细胞内多种细胞器造成损伤,并且BaP浓度越高,损伤程度越严重。     

Hox gene complexity in medaka fish may Be similar to that in pufferfish rather than zebrafish.

Changes in number and the genomic organization of Hox genes have played an important role in metazoan body-plan evolution. They make cluster(s), and in vertebrates, each cluster contains different number of Hox genes that have been classified into 13 groups. There are 39 Hox genes in four clusters on different chromosomes in the mammalian genome. In the fish, while 31 Hox genes in four clusters have been identified in pufferfish Fugu rubripes, 47 Hox genes in seven clusters exist in the zebrafish Danio rerio. To estimate the evolutionary origin of Hox organization in ray-finned fishes, we searched for Hox genes in the medaka fish Oryzias latipes, with a taxon thought to be widely separated from those of pufferfish and zebrafish. We synthesized various mixed oligonucleotides that can work as group-specific primers for PCR, then cloned and sequenced amplified fragments. Numbers of Hox genes identified in the present study were 2 for group 1, 2 for group 2, 1 for group 3, 3 for group 4, 6 for groups 5-7, 2 for group 8, 4 for group 9, 3 for group 10, 1 for group 12, and 3 for group 13. The primers specific for group 11 did not function in this study. Thus, at least 27 Hox genes are present in medaka genome, suggesting that the Hox gene complexity of the medaka genome is similar to that of the pufferfish rather than the zebrafish.