Biomarker gene response in male Medaka (Oryzias latipes) chronically exposed to silver nanoparticle.

The chronic toxicity test has been conducted for twenty-eight days to characterize the hepatic expression levels of eight stress-related genes after exposing Medaka to two doses of silver nitrate or a silver nanoparticle (Ag-NP) using real time RT-PCR analysis. This extends our previously published work to include three additional biomarkers and three later time points. In comparing with the control, the significant induction of MT and GST genes in livers of the fish exposed to 1 µg/l Ag-NPs was observed at various time points during the test period. The Orla C3-1 (Medaka) gene was slightly induced only with 1 µg/l Ag-NPs at 7-day exposure while the suppression of p53 and HSP70 was recorded in all exposures at the end of the test. The gene encoding transferrin was repressed at day 21 by both silver types and at every exposure dosage. These results revealed that the Ag-NPs increase metal detoxification, oxidative and inflammatory stress, and finally stimulate immune responses in Medaka. The conspicuous induction of choriogenin L and vitellogenin 1 in male fish exposed to Ag-NPs, especially at 7- and 21-day, compared with the exposures of AgNO(3) or control was the first attempt to examine estrogenic effects of Ag-NPs.

Construction and characterization of Japanese medaka (Oryzias latipes) hepatic cDNA library and its implementation to biomarker screening in aquatic toxicology.

To strengthen the toxicogenomic study, we constructed a library of hepatic cDNA from Japanese medaka under influence of specific chemical mediated stress responses. Gene expression profile analysis of the cDNA microarrays followed by real time RT-PCR assay were conducted to screen particular biomarkers for 17-beta estradiol (E2), nonylphenol (NP) and 2-chlorophenol (2CP). Information of 1509 high-quality ESTs including 260 new ESTs was added onto GenBank and dbEST. The ESTs were clustered and assembled into 159 contigs and 372 singletons. Among them, 128 contigs and 163 singletons (54.8%) were functionally characterized and 13 UniESTs (2.5%) were hypothetical proteins. Ontology analysis resulting in 282 UniESTs which involved with 2102 GOs and 93 sequences associated with 116 enzyme codes. For each test chemical, two specific biomarkers were selected from the gene expression profiling of microarrays. The expression patterns of the marker genes in real time PCR analysis were consistent with the regulated gene expression patterns in microarrays. The tentative biomarkers showed unique gene expression patterns depending on chemical concentration(s) and exposure duration in real time RT-PCR analysis. The analysis accomplished of the hepatic cDNA library and its information added to genetic and genomic resources could be sufficiently valuable specifically for aquatic toxicity studies.

Ginsenoside Production and Morphological Characterization of Wild Ginseng (Panax ginseng Meyer) Mutant Lines Induced by γ-irradiation ((60)Co) of Adventitious Roots.

With the purpose of improving ginsenoside content in adventitious root cultures of Korean wild ginseng (Panax ginseng Meyer), the roots were treated with different dosages of γ-ray (5, 10, 25, 50, 75, 100, and 200 Gy). The growth of adventitious roots was inhibited at over 100 Gy. The irradiated adventitious roots showed significant variation in the morphological parameters and crude saponin content at 50 to100 Gy. Therefore, four mutant cell lines out of the propagation of 35 cell lines treated with 50 Gy and 100 Gy were selected on the basis of phenotypic morphology and crude saponin contents relative to the wild type control. The contents of 7 major ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) were determined for cell lines 1 and 3 from 100 Gy and lines 2 and 4 from 50 Gy treatments. Cell line 2 showed more secondary roots, longer length and superior growth rate than the root controls in flasks and bioreactors. Cell line 1 showed larger average diameter and the growth rate in the bioreactor was comparable with that of the control but greater in the flask cultured roots. Cell lines 1 and 2, especially the former, showed much more ginsenoside contents than the control in flasks and bioreactors. Therefore, we chose cell line 1 for further study of ginsenoside contents. The crude saponin content of line 1 in flask and bioreactor cultures increased by 1.4 and 1.8-fold, respectively, compared to the control. Total contents of 7 ginsenoside types (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) increased by 1.8 and 2.3-fold, respectively compared to the control. Crude saponin and ginsenoside contents in the bioreactor culture increased by about 1.4-fold compared to that the flask culture.

Evaluation of the toxic impact of silver nanoparticles on Japanese medaka (Oryzias latipes).

The increased use of nano-sized metallic materials is likely to result in the release of these particles into the environment. It is, however, unclear if these materials are harmful to aquatic animals. Furthermore, because the dissolution of such nanomaterials will occur, it is probable that some of the adverse effects resulting will result from the dissolved metal species. In this study, therefore, we investigated the health and environmental impact of silver nanoparticles (Ag-NPs) on Japanese Medaka by studying changes in the expression of stress-related genes using real time RT-PCR analysis and compared these results with those of Medaka exposed to soluble silver ions. The stress-related genes selected here were metallothionein, HSP 70, GST, p53, CYP 1A and the transferrin gene. The expression levels of each gene were determined using two different Ag-NPs dosages and were quantified by measuring the mRNA concentrations in liver extracts with the Taqman-based Real-Time PCR method. The results suggest that these two silver forms have distinguishable toxic fingerprints between them. While the Ag-NPs led to cellular and DNA damage, as well as carcinogenic and oxidative stresses, genes related with metal detoxification/metabolism regulation and radical scavenging action were also induced. In contrast, the ionic silver led to an induction of inflammatory response and metallic detoxification processes in the liver of the exposed fish, but resulted in a lower overall stress response when compared with the Ag-NPs.

Specific responses of bacterial cells to dioxins.

Five different recombinant bioluminescent strains of Escherichia coli that contain the recA (responsive to DNA damage related stress), fabA (membrane damage), katG (oxidative damage), grpE (protein damage), and lac (constitutive expression, general toxicity) promoters fused to the bacterial lux operon from either Vibrio fischeri or Photorhabdus luminescens were used to describe the different mechanisms of toxicity that several dibenzo-p-dioxins and dibenzofurans have on bacteria, as well as to determine whether bacteria can sensitively detect the presence of these compounds. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was found to cause only DNA-related damage to bacterial cells. However, the four stress-responsive strains showed positive responses after addition of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD), whereas 2,3,7,8-tetrachlorodibenzo-p-furan (2,3,7,8-TCDF) caused only DNA. oxidative, and protein damage. However, 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD) was not found to induce any stresses tested for in this study, that is, DNA, membrane, oxidative, and protein damage, indicating that each congener might differentially interact with the cell, stimulating differential stress responses within them. By using the constitutive strain, we found that the level of cellular toxicity experienced due to the addition of these four dioxins decreased in the order of 2,3,7,8-TCDD (the most toxic). 1,2,3,4-TCDD, 2.8-DCDD, and 2,3,7,8-TCDF. The 20% effective concentration (EC20), defined in this study the concentration of chemical that causes a 20% decrease in the bioluminescence 60 min after induction, was only 0.1 microg/L for 2,3,7,8-TCDD, a value that is lower than that of the other congeners and demonstrates that 2,3,7,8-TCDD was the most toxic compound tested in this study.