Doris W.T. Au

Relationship between ultrastructural changes and EROD activities in liver of fish exposed to Benzo[a]pyrene

Laboratory experiments were carried out to investigate the relationship between hepatocytological changes and EROD activities in the demersal fish Solea ovata up to 7 days following intraperitoneal injection of 0.1, 0.5, 1, 5 and 10 mg/kg benzo[a]pyrene (B[a]P). At 1 and 5 mg/kg of B[a]P, observable cytological alterations involved increase in abundance of lipofuscin granules, peroxisomes, mitochondria, lipid droplets and lysosomes, slight proliferation of the ER system, and glycogen depletion. At a higher dose (10 mg/kg) of B[a]P, condensation of cytoplasmic content was observed. Stereological analysis also showed apparent changes in hepatic lipofuscin granules, peroxisomes, mitochondria and lipids in response to B[a]P exposure. Experimental results demonstrated a good dose-response relationship between exposure to B[a]P and changes in EROD activities. Numerical densities of hepatic lipofuscin granules and peroxisomes were significantly correlated with EROD activities (r=0.84, 0.75, respectively, P < 0.01). The possible relationship between EROD induction, increases of lipofuscin granules and peroxisomes as a result of B[a]P exposure in S. ovata is discussed. The observed link between EROD induction and hepatocytological changes in response to toxicant exposure provides a more meaningful interpretation of EROD induction in ecotoxicological studies.

Reproductive impairment of sea urchins upon chronic exposure to cadmium. Part I: Effects on gamete quality.

Successful reproduction is a determining factor for species survival. Pollution may impair reproductive success of adults through effects on gamete quality. Reproductive impairment of the sea urchin Anthocidaris crassispina upon chronic (4 weeks) exposure to 0.01 and 0.1 mg l-1 Cd2+ was investigated. Criteria used for assessing gamete quality included sperm motility, egg morphology, fertilization rate and dynamics of first cleavage. A dose-response relationship was found between Cd2+ levels and changes in sperm motility, and percentage fertilization. Sperm motility, measured by computer-assisted sperm analysis, indicated that percent motile sperm, velocities, and percent sperm with normal trajectory were significantly affected by chronic exposure to > or = 0.1 mg l-1 Cd2+. A decline in sperm motility was also accompanied by a decrease in fertilization success of sea urchin sperm. Width/height ratio of sea urchin eggs was not affected by cadmium, but larger egg sizes were found when sea urchins were exposed to 0.1 mg l-1 Cd2+. Male sea urchins exposed to Cd2+ produced poorer quality sperm, as indicated by a lower percent fertilization and lower cleavage rate, implying that male sea urchins were more sensitive than females to chronic Cd2+ exposure. Results of the present study provide an explanation of reproductive impairment in marine invertebrates upon chronic exposure to Cd2+.

The marine medaka Oryzias melastigma – A potential marine fish model for innate immune study

The objective of this study is to develop the marine medaka Oryzias melastigma as a potential marine fish model for innate immune and immunotoxicological studies. Hepcidin plays an important role in innate immune system. Two hepcidin genes (OM-hep1 and OM-hep2) were identified and characterized in the O. melastigma, which were highly conserved with other reported hepcidins. During embryogenesis, significant elevation of OM-hep1 and OM-hep2 transcripts were coincided with liver development in the embryos. In adult medaka, differential tissue expressions of both hepcidin transcripts were evident: high in liver, moderate in spleen and low in non-immune tissues. After bacterial challenge, the two hepcidin mRNAs were rapidly and remarkably induced in liver and spleen, suggesting the two OM-hepcidins in O. melastigma play a complementary role in innate defense. Gender difference in time of induction and extent of the two hepcidin mRNAs elevation in infected O. melastigma should be considered in immunotoxicological studies.

Effect of osmotic shrinkage and hormones on the expression of Na+/H+ exchanger-1, Na+/K+/2Cl- cotransporter and Na+/K+ -ATPase in gill pavement cells of freshwater adapted Japanese eel, Anguilla japonica.

SUMMARY It is well-known that gill epithelial cells are important in fish osmoregulation. However, studies on the effect of osmotic stress on the direct cellular responses of the gill epithelial cells are limited. In this paper, we aimed to determine the effects of osmotic hypertonicity, hormones and cellular signaling molecules on the expression of ion transporters in the cultured primary freshwater pavement cells (PVCs), prepared from freshwater-adapted eels (Anguilla japonica). Our data demonstrated that the hypertonic (500 mOsmol l–1) treatment of the isolated PVCs induced cell shrinkage, followed by regulatory volume increase (RVI). Application of blockers (i.e. ouabain, bumetanide and EIPA) demonstrated that Na+/K+-ATPase, Na+/K+/2Cl– cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) were involved in RVI. Western blot analysis of the hypertonic-treated cells revealed a significant induction of NHE-1, NKCC and, α and β subunits of Na+/K+-ATPase. In nonshrunken cultured PVCs, we found that dexamethasone and dibutyryl cAMP treatments significantly stimulated the expression levels of the three ion transporters. Both prolactin and insulin-like growth factor-1, can only induce the expression of NKCC. The effect of thyroid hormone (T3) and dibutyryl cGMP was negligible. In this study, the induction of ion transporter expression was found to be post-transcriptionally regulated as no significant change in mRNA levels was detected. This observation implies that the regulation is rapid and is probably induced via nongenomic actions.

Nuage constituents arising from mitochondria: is it possible?

An ultrastructural study of nuage–mitochondria complexes in spermatogonia of the sea urchin, Anthocidaris crassispina, was carried out. Release of mitochondrial contents into the cytoplasm was observed. The mitochondrial derivatives persisted as cristae‐containing globules of friable material that subsequently contacted and integrated with nuage. The present ultrastructural findings agree with the results of other researchers who proposed that germ plasm substance probably produced by the nucleus is supplemented by the mitochondrial genome.

Time‐Dependent transcriptional profiles of genes of the hypothalamic‐pituitary‐gonadal axis in medaka (Oryzias latipes) exposed to fadrozole and 17β‐trenbolone

Both the anabolic androgen 17beta-trenbolone (TRB) and the aromatase inhibitor fadrozole (FAD) can cause decreased plasma concentrations of estrogen (E2) and reduce fecundity of fish. However, the underlying mechanisms and the molecular pathways involved are largely unknown. The present study was designed to assess time-dependent effects of FAD and TRB on the transcriptional responses of the hypothalamic-pituitary-gonadal (HPG) axis of Japanese medaka (Oryzias latipes). Fourteen-week-old Japanese medaka were exposed to 50 microg FAD/L or 2 microg TRB/L in a 7-d static renewal test, and the expression profiles of 36 HPG axis genes were measured by means of a medaka HPG real-time reverse-transcription polymerase chain reaction array after 8 h, 32 h, or 7 d of exposure. Exposure to TRB or FAD caused lesser fecundity of Japanese medaka and down-regulated transcription of vitellogenin and choriogenin (CHG) gene expression in the liver of females. Exposure to FAD for 8 h resulted in an 8-fold and 71-fold down-regulation of expression of estrogen receptor alpha and choriogenin L (CHG L), respectively, in female liver. 17beta-Trenbolone caused similar down-regulation of these genes, but the effects were not observed until 32 h of exposure. These results support the hypothesis that FAD reduces plasma E2 more quickly by inhibiting aromatase enzyme activity than does TRB, which inhibits the production of the E2 precursor testosterone. Exposure to FAD and TRB resulted in rapid (after 8 h) down-regulation of luteinizing hormone receptor and low-density-lipoprotein receptor in the testis to compensate for excessive androgen levels. Overall, the molecular responses observed in the present study differentiate the mechanisms of the reduced fecundity by TRB and FAD.

Occurrence and fate of pharmaceuticals and personal care products in drinking water in southern China

Occurrence and fate of pharmaceuticals and personal care products (PPCPs) in drinking water was investigated in southern China. Fifteen and twelve PPCPs were detected with concentrations of 0-36 ng L(-1) in source water and of 0-20 ng L(-1) in treated water, respectively. Four PPCPs were detected with concentrations of approximately 1 ng L(-1) in drinking water of distribution network. Conventional water treatment processes removed the types and average concentrations of PPCPs by 30% and above 50%, respectively. Advanced water treatment processes were more efficient in the removal of most PPCPs, with the types and concentrations reduced by 50% and approximately 90%, respectively. Molecular properties of PPCPs had an important influence on their behaviors during water treatment. pK(a) (acidity coefficient) and K(oc) (organic carbon partition coefficient) of PPCPs appeared to have a combined effect on PPCPs removal during coagulation and oxidation. Adsorption and biodegradation were two possible mechanisms responsible for PPCPs removal during sand filtration.

Hollow superparamagnetic iron oxide nanoshells as a hydrophobic anticancer drug carrier: intracelluar pH-dependent drug release and enhanced cytotoxicity

With curcumin and doxorubicin (DOX) base as model drugs, intracellular delivery of hydrophobic anticancer drugs by hollow structured superparamagnetic iron oxide (SPIO) nanoshells (hydrodynamic diameter: 191.9 ± 2.6 nm) was studied in glioblastoma U-87 MG cells. SPIO nanoshell-based encapsulation provided a stable aqueous dispersion of the curcumin. After the SPIO nanoshells were internalized by U-87 MG cells, they localized at the acidic compartments of endosomes and lysosomes. In endosome/lysosome-mimicking buffers with a pH of 4.5-5.5, pH-dependent drug release was observed from curcumin or DOX loaded SPIO nanoshells (curcumin/SPIO or DOX/SPIO). Compared with the free drug, the intracellular curcumin content delivered via curcumin/SPIO was 30 fold higher. Increased intracellular drug content for DOX base delivered via DOX/SPIO was also confirmed, along with a fast intracellular DOX release that was attributed to its protonation in the acidic environment. DOX/SPIO enhanced caspase-3 activity by twofold compared with free DOX base. The concentration that induced 50% cytotoxic effect (CC(50)) was 0.05 ± 0.03 μg ml(-1) for DOX/SPIO, while it was 0.13 ± 0.02 μg ml(-1) for free DOX base. These results suggested SPIO nanoshells might be a promising intracellular carrier for hydrophobic anticancer drugs.

Evaluation of biomarker potential of cytochrome P450 1A (CYP1A) gene in the marine medaka, Oryzias melastigma exposed to water-accommodated fractions (WAFs) of Iranian crude oil

CYP1A is involved in the metabolism of diverse chemicals, including polycyclic aromatic hydrocarbons and alkylated-PAHs, as a first line of detoxification mechanism. First, we identified and characterized the CYP1A gene from the marine medaka, Oryzias melastigma. O. melastigma CYP1A (Om-CYP1A) showed a high similarity of motifs/domains compared to those of vertebrates in their amino acid sequences. To check whether the Om-CYP1A would be inducible, we tested two strong CYP1A inducers, β-naphthoflavone (β-NF) and benzo[α]pyrene (B[α]P), and observed concentration-dependent transient expression on transcripts of Om-CYP1A for 96 h over a wide range of concentrations. Om-CYP1A mRNA level was significantly increased in exposure to different concentrations of β-NF and B[α]P, and its expression was highly transcribed within 12 h upon the exposure to low concentrations of both chemicals. Inducible transcript profiles revealed that Om-CYP1A would be associated with the toxicant metabolism via AhREs/DREs/XREs in its promoter region. To uncover the effects of the water-accommodated fraction (WAF) of crude oil on transcripts of Om-CYP1A, we measured mRNA expression of Om-CYP1A towards different concentrations of WAF for 24h. As a result, WAF exposure significantly increased Om-CYP1A transcripts at all concentrations as well as during time-course experiments for 96 h. In this paper, we demonstrated that WAF would trigger up-regulation of the CYP1A gene that would be associated with the initiation of the cellular defense systems. This finding provides a better understanding of the molecular mechanism of cellular protection particularly that involved in the WAF-mediated cellular response in O. melastigma.

Tissue-specific transcriptome assemblies of the marine medaka Oryzias melastigma and comparative analysis with the freshwater medaka Oryzias latipes

BackgroundThe marine medaka Oryzias melastigma has been demonstrated as a novel model for marine ecotoxicological studies. However, the lack of genome and transcriptome reference has largely restricted the use of O. melastigma in the assessment of in vivo molecular responses to environmental stresses and the analysis of biological toxicity in the marine environment. Although O. melastigma is believed to be phylogenetically closely related to Oryzias latipes, the divergence between these two species is still largely unknown. Using Illumina high-throughput RNA sequencing followed by de novo assembly and comprehensive gene annotation, we provided transcriptomic resources for the brain, liver, ovary and testis of O. melastigma. We also investigated the possible extent of divergence between O. melastigma and O. latipes at the transcriptome level.ResultsMore than 14,000 transcripts across brain, liver, ovary and testis in marine medaka were annotated, of which 5880 transcripts were orthologous between O. melastigma and O. latipes. Tissue-enriched genes were identified in O. melastigma, and Gene Ontology analysis demonstrated the functional specificity of the annotated genes in respective tissue. Lastly, the identification of marine medaka-enriched transcripts suggested the necessity of generating transcriptome dataset of O. melastigma.ConclusionsOrthologous transcripts between O. melastigma and O. latipes, tissue-enriched genes and O. melastigma-enriched transcripts were identified. Genome-wide expression studies of marine medaka require an assembled transcriptome, and this sequencing effort has generated a valuable resource of coding DNA for a non-model species. This transcriptome resource will aid future studies assessing in vivo molecular responses to environmental stresses and those analyzing biological toxicity in the marine environment.