OMICS(genomics, proteomics, metabolomics)technology is being applied to biological studies from prokaryote to human. In my laboratory, we are trying to combine OMICS technologies to environmental monitoring using yeast cells, medaka, rice and some kinds of animals. In this report, I would like to focus on OMICS as the tools of environmental science. For genomics studies, we use DNA microarray and this is now commercially available. DNA microarrays have enabled genome-wide analyses of cellular responses at the transcriptional level. This technology provides a great opportunity for bioassays of chemical and environmental toxicity because it can provide an overview of thousands of genes at the same time and shed light on how to investigate toxicological problems. For proteomics studies, classical two-dimensional electrophoresis and peptide sequencer or mass spectrometry were used for monitoring stress induced and modified proteins. However, protein turnover ratio especially degradation rate was slow in stressed cells and the induction levels of proteins do not always reflect the timely status of physiology. The role of proteomics must be the contribution to modified protein analysis. For metabolomics studies, NMR, LC/MS, and CE/MS are candidate for the separation and identification of metabolites. The most advanced point of metabolomics is the fact that we, human, shares metabolite with almost all organisms.
The future of OMICS technology depends how combine genomics, proteomics and metabolomics, how scientist open their results, and how we apply to organism living in the environment.

View full abstract

¹H-NMR based metabolomics was applied in carp plasma orally exposed to heavy A oil (1% in fish food) for 2 weeks. Plasma samples were collected at 0 (immediately before) , 1, and 2-week after exposure to heavy oil. ¹H-NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by heavy oil toxicity. Concentrations of total polycyclic aromatic hydrocarbons (ΣPAHs) composed heavy oil increased from 51.5 ng/g-dry to 568 ng/g-dry during exposure period, and carp certainly absorbed heavy oil. Metabolic effects in plasma from exposed carp indicated that lipids had been increasing during exposure period. In addition, plasma spectra showed increases of isoleucine, leucine, alanine, proline, tyrosine, histidine, phenylalanine, 3-hydroxybutyrate, glucose, and myoinositol from 0 to 1-week. These changes could be induced to the hepatic and renal toxicity by exposure heavy oil. These metabolites except for isoleucine, alanine, and 3-hydroxybutyrate decreased at 2-week, and liver and kidney tended to recover from the toxicity damages.

View full abstract

Recently, concern over the influence of manufactured nanoparticles on human health has risen due to advances in the development of nanotechnology. We are interested in the influence of nanoparticles on the pulmonary system at a molecular level. In this study, gene expression profiling of the rat lung after intratracheal instillation or whole-body inhalation exposure to ultrafine nickel oxide (Uf-NiO) particles as a positive control of manufactured nanoparticles such as titanium dioxide, C60 fullerene, carbon nanotube were employed to gain insights into these molecular events. In response to Uf-NiO exposure, there were a large number of highly up-regulated genes associated with the inflammatory response and Mmp12 encoding macrophage metalloelastase 12. These results suggest that Uf-NiO particles lead to acute inflammation immediately after intratracheal instillation or for the inhalation exposure period, and the damaged tissues were repaired in a timedependent manner. Analysis by inductively coupled plasma mass spectrometry and transmission electron microscope revealed that the Uf-NiO particles began to be cleared from the lungs immediately after treatment, and that low levels of the particles were present following exposure. These observations were consistent in gene expression profiling. We conclude that gene expression analysis using DNA microarrays can be extremely useful in assessing the influence of utrafine particles on biological systems.

View full abstract

We have analyzed the gene expression profiles in the liver of wild common cormorants (Phalacrocorax carbo) using microarray platform, and have predicted the potential toxic effects of persistent organic pollutants (POPs) and emerging POPs. However, when applying microarray technology to wild animals, it is quite difficult to clarify the cause-andeffect relationship between accumulated contaminants and gene expression alterations. Therefore, in the present study, we isolated liver cells from wild cormorant embryos, and the cells were cultured and treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3′,4,4&prime,5-pentachlorobiphenyl (PCB126). The gene expression profiles in the cultured cells were monitored by microarray analysis, and responses to dioxins were then compared between livers (in vivo) and cultured cells (in vitro) from wild cormorants. The cultured liver cells clearly exhibited responses to the exposure of TCDD or PCB126 including inductions of cytochrome P450 1A genes. Whereas fifteen genes on the microarray showed similar effects between in vivo and in vitro tests, indicating that these genes might be affected directly by dioxin exposure, the responses of other genes were different between wild cormorant livers and cultured cells. Therefore, we compared the ‘biological process’ gene ontology (GO) terms of dioxin-responsive genes between in vivo and in vitro tests. The most of annotated GO terms were shared in the livers and cultured cells, implying that the effects by dioxins were similar between both groups. Thus, gene expression profiling in the cells isolated from cormorants might be useful for evaluating chemical effects on the wild population.

View full abstract

Metabolomic approach to ecological risk assessments of chemicals(ecotoxicometabolomics) has great potential for facilitating a better understanding of toxicity pathways or mode of actions (MOAs). The purpose of this study is to establish a system that can detect the MOAs by capturing the change of etabolome in green algae (Pseudokirchneriella subcapitata), commonly used for the ecotoxicological test as a model organism in freshwater, treated with the bioactive compounds. P.subcapitata were cultured in accordance with the OECD test guideline No.201. Exponentially growing P.subcapitata was exposed to the bioactive compounds, as herbicides, with each different MOA at 50%-effective concentration (ErC50) level. The metabolite was extracted from the algae using d4-methanol, hence metabolome was measured by ¹H-NMR. The ¹H-NMR spectra were analyzed by pattern recognition method, known as principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA). The score plot of PCA showed clear classification between each group treated with and without chemicals. In addition, increases and decreases of the fraction that considerably contributed to separation of each PCA were also reflected on the spectrum of ¹H-NMR. Moreover, the results of SIMCA gave the good presentation of classification reflecting the difference of MOA between with and without exposure to chemicals. It was suggested that MOA for bioactive compounds would be classified by ecotoxicometabolomic approach using ¹H-NMR measurements coupled with pattern recognition method

View full abstract

Ecotoxicogenomics has emerged as an attractive approach for clarifying critical molecular events altered by ecotoxicants. The aim of the present study was to develop a prediction method for a narcotic mode of toxic action (i.e., baseline toxicity) of chemicals by analyzing the differentially expressed genes in medaka (Oryzias latipes) following exposure. Male medaka were exposed to 15 chemicals including narcotic and reactive compounds for one hour. After exposure, the gene expression profiles of the whole bodies were analyzed using a custom microarray with 36,398 nonredundant transcripts. A total of 576 genes differentially expressed between medaka exposed to narcotic and reactive chemicals were statistically selected. Then, a gene network analysis was performed for their human orthologs in order to find gene sets discriminating between these two toxicant classes. The highest ranked network of genes contained 22 genes, which are involved in calpain-mediated signaling. Subsequently, support vector machine was employed to develop prediction model of baseline toxicity. The overall prediction accuracy by means of leave-one-out crossvalidation was 86.7%. These results indicate that one-hour exposure assays for ecotoxicant classification using transcript profiling in medaka have great promise.

View full abstract

Ascidians are marine invertebrate chordates. We have developed a large-scale oligo DNA microarray of the ascidian Ciona intestinalis. We initially obtained expression patterns of 10,000 genes of this animal during its life cycle. We have now constructed a novel risk assessment system for marine chemical pollutants using ascidian DNA microarrays. First, we identify genes up- or down-regulated after exposing ascidians to pollutants. Second, we estimate the effects of the pollutants on the ascidians using array data and information about the life stages highly expressing those genes. Third, to verify the estimation, we investigate the inhibitory effects of pollutants on embryogenesis and metamorphosis in ascidians. From DNA microarray data, we estimated that organotin compounds might affect embryogenesis and metamorphosis in ascidians. We performed bioassay using embryo and larvae and found that a low concentration of the chemicals completely inhibited ascidian embryogenesis and metamorphosis. Our new risk assessment system will be a valuable tool for monitoring marine chemical pollutants.

View full abstract

Bioaccumulation and hepatic EROD induction were investigated in the red sea bream and Java medaka exposed to dietary-borne PAHs and compared to a previous water-borne exposure.
Of the four test PAHs, phenanthrene, pyrene, and chrysene were detected in the red sea bream liver and muscles, benzo[a]pyrene was detected only in the muscles. All accumulations decreased with time. In the water-borne exposure for red sea bream, only phenanthrene and pyrene were accumulated and concentrations increased with exposure duration. The Java medaka accumulated all the 4 PAHs as in the water-borne exposure with the same trend.
Hepatic EROD activity in both species increased 3-100 folds over control. Red sea bream inductions were comparable to the water-borne inductions, and they persisted during the depuration period. Java medaka inductions were not significantly different from the water-borne exposure and they declined in the depuration.
These results suggest that high molecular weight PAHs like chrysene and benzo[a]pyrene can be accumulated by red sea bream through the food chain, but the low molecular weight PAHs will be readily accumulated through water. Irrespective of the uptake route, the Java medaka can accumulate all the four PAHs. It seems PAH uptake route did not influence hepatic EROD induction.

View full abstract

Concentrations of 21 trace elements (V, Cr, Mn, Co, Cu, Zn, Se, Rb, Sr, Mo, Ag, Cd, In, Sn, Sb, Cs, Ba, Hg, Tl, Pb and Bi) were determined in the liver and muscle of Baikal seals (Pusa sibirica) (n = 44) collected from Lake Baikal, Russia in 2005. Most of the elements, such as V, Mn, Cu, Zn, Se, Hg and Mo, showed higher concentrations in liver than in muscle. However, Cs was found at higher levels in muscle than in liver. Liver and muscle concentrations of essential elements Cu and Zn were higher than other essential elements in Baikal seals. On the other hand the levels of non-essential elements Cd and Hg were comparable to or lower than the corresponding levels in other seal species. Significant age dependent accumulation was observed for Mn, Cu, Sr, Cd and Hg in liver and muscle, V, Se, Mo and Ba in liver, and Zn in muscle. Significant correlations were observed between concentrations of the following trace elements V-Cd, Mn-Cu, Mn-Cd, Cu-Sr, Cu-Cd, Cu-Hg, Rb-Cs, Sr-Ba, Sb-Tl, Ba-Pb and Tl-Bi in both liver and muscle. Generally, the molar ratio between Se and Hg is known to be 1:1 in marine mammals which show high levels of Se and Hg, however no such pattern was observed in the present study due to the low level of these elements. Concentrations of V, Cr, Mn, Co, Cu, Zn, Se, Sr, Mo, Ag, Cd, Sb, Cs, Hg, and Tl in Baikal seals collected in 2005 were comparable to those in the seals collected in 1992. On the other hand, concentrations of Rb were significantly higher in 2005 samples, suggesting that environmental contamination by Rb in Lake Baikal seems to have increased during the last decade.

View full abstract

In this study, we investigated the toxicity of bisphenol compounds (BPs), namely, bisphenol A (BPA), bisphenol B (BPB), bisphenol E (BPE), bisphenol F (BPF) and bisphenol P (BPP) to the early life stages of medaka, and their estrogenic activity in vitro based on chemiluminescent signal intensity. The 14-d LC₅₀ values of BPs on embryos less than 24-h post-fertilization were estimated in the range of 2800 to 28600 μg/L(BPA: 14800 μg/L, BPB: 7400 μg/L, BPE: 26000 μg/L, BPF: 28600 μg/L and BPP: 2800 μ g/L). In addition, the lowest-observed-effect concentrations of hatchability after 14-d BPs exposure were estimated as follows: BPA: 12500 μg/L, BPB: 8890 μg/L, BPE: 26670 μ g/L, BPF: 17780 μg/L and BPP: 1780 μg/L. Moreover, the time to hatching of fertilized eggs exposed to BPs for 14 days was significantly delayed at a BPF dose of 17780 μg/L. The 96-h LC₅₀ values of BPs on 24-h-old larvae were estimated in the range between 6100 and 13900 μg/L(BPA: 13900 μg/L, BPB: 6100 μg/L, BPE: 13900 μg/L, BPF: 13300 μg/L and BPP: 2300 μg/L). On the other hand, the estrogenic activities of BPs for medaka estrogen receptor α were estimated as BPA: 0.06, BPB: 0.10, BPE: 0.03, BPF: 0.13 and BPP: 0.05 compared with estradiol-17β: 100 in the yeast two-hybrid assay. These results suggest that BPB and BPP have high toxicity to the early life stages of medaka and that the in vitro estrogenic activities of BPB and BPF were higher than that of BPA.

View full abstract

Calcineurin (CN) is a Ca²⁺/calmodulin-dependent protein serine/threonine phosphatase that plays a pivotal role in a variety of cellular functions, such as the nerve and immune systems in our body. It has been shown that CN can be stimulated by divalent ions, such as Mn²⁺ and Ni²⁺, and inhibited by Zn²⁺. In this study, when we tested the effects of a chemical compound on some enzyme activities, we happened to find that elutants form the O-ring of a plastic tube used for the storage of the chemical compound specifically inhibits CN activity in vitro. We tried to elute the inhibitors with several solvents, partially purified the inhibitors, which were hydrophilic and hydrophobic ones, and found that one of the CN inhibitors could be Zn²⁺. Although the hydrophobic inhibitor(s) of CN eluted with ethanol have not been identified, the inhibitors were not plasticizers, such as potassium hydrogen phthalate and diethyl phthalate. Moreover, we found that some ordinary plastic and rubber products contain CN inhibitor(s). These results indicate that CN inhibitors eluted from ordinary plastic goods may disturb reliability and fidelity of our experimental data and/or be toxic to our body by disturbing our nerve and immune systems.

View full abstract

Aquatic toxicity of effluents from five pulp-paper mill was evaluated in an acute toxicity study employing Daphnia bioassay. Toxicity test results revealed 24-h EC₅₀ values > 100, 87.83, 85.69, 78.15, 87.53%, and 48-h EC₅₀ values > 100, 82.84, 46.44, 69.55 and 77.87% for five pulp-paper mill(PP1-PP5), respectively. Toxicity test results showed linear relationship with high degree of confidence(≥ 0.75 R2 ≥ 0.99) between immobility and test concentrations. The general criteria of toxicity classification showed that 4 out of 5 pulp-paper mill effluents tested were minor acutely toxic having 48-h EC₅₀ in between > 46-100% and 1 was not acutely toxic. Toxicity classification based on toxic unit revealed that 4 out of 5 pulp-paper mill effluents were toxic.

View full abstract

Culture and reproduction testing of E. fetida were conducted successfully with a dry powdered food of rolled barley and spinach (4:1) instead of the TG-222-recommended cow or horse manure. Controls had produced 158.5±27.4(n=4) juveniles by the test's end. We propose that number of juveniles produced by 10 control adults in the TG- 222 validity criteria be increased from more than 30 to more than 60 considering the reproduction potential of this species. The amount of food given initially to 10 adults or to the juveniles produced by these adults should be less than the TG-222-prescribed 5 g. A first feeding of 1 g (for adults) or 0.8 g (for juveniles) minimized fungal growth. The amount and timing of second and subsequent feeds should be determined from the amount of food uneaten. Hatchability of eggs in a cocoon and/or number of eggs (as juveniles)/cocoon can be calculated from additional sampling of vacant cocoons at the end of test, 4 weeks after adult removal. Toxicity data from our modified test were (based on production of juveniles) :LOEC, 4 mg/kg; NOEC, 2 mg/kg; and EC₅₀, 5.6 mg/kg(4.7- 7.7 C.I. mg/kg)(dry wt. soil, nominal concentrations).

View full abstract