Te-Hao Chen

Chronic exposure of 2,2′,4,4′-tetrabromodiphenyl ether (PBDE-47) alters locomotion behavior in juvenile zebrafish (Danio rerio)

In the present study, we used zebrafish (Danio rerio) as a model to address possible effects of chronic exposure of polybrominated diphenyl ether (PBDE) flame retardants on locomotion behavior, body size, and gonad development in fish. Zebrafish were fed food dosed with PBDE-47 (control, solvent control, low, medium, and high dose groups) from 21 days post hatch (dph) to 90 dph. Fish locomotion parameters, including maximum swimming speed, total distance moved, and percent time active, were assessed using a video-based animal movement analysis system. At the end of the exposure, all fish were euthanized for length and weight measurement, and then subjected to either whole fish histological analysis or tissue PBDE-47 measurement. Survival, body size, and gonad histology were similar between the five groups. However, both total swimming distance and percent time active were negatively correlated with tissue PBDE-47 concentration and were significantly lower in the high dose group. Tissue levels of PBDE-47 in the exposed fish were comparable to that reported in previous field studies. In summary, this study showed that developmental exposure of PBDE-47 at an ecologically relevant level altered locomotion behavior without affecting body size or gonad development of zebrafish.

Developmental exposures to ethanol or dimethylsulfoxide at low concentrations alter locomotor activity in larval zebrafish: implications for behavioral toxicity bioassays.

Ethanol and dimethylsulfoxide (DMSO) are commonly used as carrier solvents for lipophilic chemicals in aquatic toxicity bioassays. However, very little information has been reported on the behavioral effects of these solvents. In this study, we examined the effects of ethanol and DMSO on development and locomotor activity by a zebrafish embryo-larval bioassay. The zebrafish were exposed to different concentrations (control, 0.01, 0.1, and 1%) of ethanol or DMSO from blastula stage to 144 hour-post-fertilization (hpf). Hatchability, survival, and abnormalities were monitored every 12h, and locomotor activity of the larvae was analyzed at 144 hpf. Hatchability was not affected by the ethanol or DMSO treatments. No effect on survival was observed except the 1% ethanol group suffered 89% mortality during 108-120 hpf. No developmental defects were observed in any of the solvents at the 0.01 and 0.1% concentrations, but significantly higher deformity rates occurred with 1% ethanol and DMSO groups. Hyperactivity and less tortuous swimming paths were observed in all ethanol and DMSO concentrations. Based on this study, we suggest that data of behavioral toxicity bioassays using ethanol or DMSO as carrier solvents should be interpreted cautiously, because the solvents at low concentrations could alter locomotor activity of larval zebrafish without causing any observable developmental defects.

Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish

Background Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene. Methodology/Principal Findings Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1)cy18 displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1)cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1)cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1)cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1)cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues. Conclusion/Significance Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity.

Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

Background Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. Methodology/Principal Findings This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR+ fluorescent signaling. Conclusion/Significance The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.

Assessing the effects of polychlorinated biphenyls (Aroclor 1254) on a scleractinian coral (Stylophora pistillata) at organism, physiological, and molecular levels.

Polychlorinated biphenyls (PCBs) are a group of widespread contaminants, and accumulation of PCBs has been observed in corals in the field. However, the toxic effects of PCBs on corals have not been investigated. In this study, we tested short and long term toxicity of Aroclor 1254, a commercial PCB mixture, on the scleractinian coral Stylophora pistillata. Coral nubbins were incubated in either control seawater or seawater dosed with PCBs (approximately 300ng/L) for 96h. The effect of PCB exposure on coral gene expression at 4h post exposure was tested with the suppression subtractive hybridization (SSH) and quantitative PCR methods. Photosystem II activity of the zooxanthellae was measured at 96h. After the exposure, nubbins were moved into clean seawater and their survival and growth were observed for another 50 days. All nubbins survived during the exposure and the following 50-d recovery period. Photosystem II activity and coral growth were not affected by PCB exposure in this study. Fifty-four clones were sequenced for gene expression analysis, and 15% of these sequences were identified, including genes involved in general stress response, peptide metabolism, cellular receptor, cytoskeleton organization, membrane trafficking, and oxidative stress response. However, the quantitative PCR did not show significant difference in the five selected genes. In conclusion, acute exposure of S. pistillata to Aroclor 1254 at 300ng/L did not affect coral survival, photosynthesis or growth but may alter the expression of certain genes involved in various important cellular functions. The nubbin technique proved to be an efficient approach to simultaneously characterize the impact of PCBs on the corals at multiple biological levels.

Endocrine disrupting effects of domestic wastewater on reproduction, sexual behavior, and gene expression in the brackish medaka Oryzias melastigma

The objective of this study was to investigate the endocrine disrupting effects of domestic wastewater on fish using the brackish medaka Oryzias melastigma as the animal model. Estuarine water samples were collected from Sihchong Creek and Baoli Creek estuaries, Taiwan, in March of 2012 to assess the whole effluent toxicity (WET) of domestic wastewater produced by the local residents and tourists. Chemical analysis detected various pharmaceuticals and personal care products (PPCPs) in the field water samples. Some of these PPCPs are endocrine disrupting chemicals. In the laboratory-based bioassay, breeding pairs were exposed to the water samples (Sihchong, Baoli, and control) for 21 days. Cumulative number of eggs spawned was significantly higher in the Sihchong group. While fish swimming activity was not affected, sexual behavior of the male fish was significantly induced in both Sihchong and Baoli groups. Male and female gonad histology was not affected. Expression level of biomarker genes CYP1A1, HSP70, and VTG was significantly induced in the Sihchong group. This study indicates that the mixture of contaminants contained in the estuarine water may cause endocrine disrupting effects in fish.

Effect of the UV-filter benzophenone-3 on intra-colonial social behaviors of the false clown anemonefish (Amphiprion ocellaris)

The UV-filter benzophenone-3 (BP-3) is widely used and is environmentally stable, lipophilic, and bioaccumulative. Previous in vitro and in vivo studies have shown that BP-3 can cause endocrine disrupting effects. However, little information is available on its ecotoxicity on coral reef fish. Agonistic behavior, which is regulated by the endocrine system, is crucial to the social structure of some coral reef fish species. Endocrine disruptors may disturb fish agonistic behavior and social interactions. In this study, we tested whether chronic BP-3 exposure can affect social behaviors in coral reef fish. Juvenile false clown anemonefish (Amphiprion ocellaris) were exposed to BP-3 via diet (0 and 1000 ng/g food) for 90 d. Through the experiment, each tank was videotaped and behavioral indicators of social status, including threatening, attacking, and submissive behaviors were quantitatively analyzed from the videos. Survival and growth were not affected by the BP-3 exposure except that the body weight of the dominant fish was higher in the BP-3 group. Social rankings were not changed by BP-3. Intra-colonial social behaviors were significantly affected only by rank but not by the BP-3 exposure. Our results suggest that BP-3 at environmental levels may not cause significant harm to social behavior of coral reef fish. However, more research is needed to better understand the behavioral effects of BP-3 in fish.

Persistent organic pollutants in Antarctic notothenioid fish and invertebrates associated with trophic levels

Notothenioid fish and invertebrate samples from Antarctica were collected in the austral summer of 2009, and analyzed for persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OCPs), and polybrominated diphenylethers (PBDEs), as well as δ13C and δ15N stable isotopes for trophic level determination. In this study, the POP levels in the Antarctic biota samples were found to be ranked in the following order: OCPs > PAHs >> PBDEs. The POP levels in notothenioid fish and krill correlate to trophic levels; however, the POP concentrations in intertidal benthic invertebrates are higher than in notothenioid fish implying that specific biogeochemical factors may affect bioaccumulation in the Antarctica ecosystem. Biomagnification of POPs may have a smaller role than bioconcentration in Antarctica environment. In addition to the source, transport, exposure, and absorption for each group of POPs in the short food chain in Antarctica, the biological variation among species, interaction habitats, diet and metabolism are also factors for future studies on contaminant bioaccumulation.

Concentration of Polycyclic Aromatic Hydrocarbons in the Seawater of Houwan Affected by the Grounding of the Chemical Tanker W-O Budmo

On 8 August 2009, a German chemical tanker known as the W-O Budmo ran aground near Checheng, Pingtung, spilling oil into the Houwan area. Polycyclic aromatic hydrocarbons (PAHs), which have acute toxicity and sublethal effects to aquatic organisms, are major components found in oil and therefore there was great cause for concern. Concentrations of PAHs were monitored in seawater and sediments at several sampling sites near Houwan to evaluate the potential environmental impact caused by this incident. Two sampling sites (C and D) were established near the grounding location to capture the immediate spread of PAHs. Two other sites (B and E) were established 0.5 km north and south of the tankers position to monitor the potential spread of pollutants. Site A was the water inlet of the National Museum of Marine Biology and Aquarium (NMMBA) to monitor seawater quality input to NMMBA. The reference station, site F, was at Wanliton. Samples were collected from the surface seawater (＜5 m) of Houwan from 10 August to 29 December 2009. The results indicated that the total PAH concentrations gradually decreased to the background levels at the sites where the W-O Budmo ran aground. Principal component analysis (PCA) of the seawater samples in this study was used to compare the PAH compound patterns of the oil spilled from the W-O Budmo. The results indicated that seawater at sites B, C, and D were affected by oil spilled from the W-O Budmo during the first week of the incident. One week after the incident, the PAH compound patterns at sites B, C, and D were similar to those of other sites, and were within environmental background levels from a previous study within the study area prior to the oil spill. Total PAH concentrations measured in surface sediment samples collected from inter-tidal areas were much lower than that previously reported in the same area, indicating that no oil residue was transported to the sediments in the inter-tidal areas. This study suggests that total PAH concentrations in the Houwan coastal area were not seriously affected by the grounding of the chemical tanker W-O Budmo.

Extraction of Polycyclic Aromatic Hydrocarbons from: A Sediments Comparison between Accelerated Solvent Extraction and Soxhlet Extraction Techniques

The methods of simultaneous extraction of polycyclic aromatic hydrocarbons (PAHs) from field sediments using Soxhlet extraction and accelerated solvent extraction (ASE) were established, and the extraction efficiencies were systemically compared from procedural blank, limits of detection, spiked method recovery, and other factors. The results showed that the values obtained in this study were comparable with the values reported by other studies. In the respects of method recovery and analysis parameter there were no significant differences between ASE and Soxhlet methods for the extraction of PAHs. In this study, under the ASE operational parameters of 100 ℃, 1500 psi, and 5 minutes, and extraction solvents of acetone and hexane, it receives a reliable extraction effectiveness compared to Soxhlet extraction. Overall, considering solvent consumption and extraction time, ASE is preferable to Soxhlet extraction.