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A novel aquaporin 3 in killifish (Fundulus heteroclitus) is not an arsenic channel.

ABSTRACT: The Atlantic killifish (Fundulus heteroclitus) is a model environmental organism that has an extremely low assimilation rate of environmental arsenic. As a first step in elucidating the mechanism behind this phenomenon, we used quantitative real-time PCR to identify aquaglyceroporins (AQPs), which are arsenite transporters, in the killifish gill. A novel homolog killifish AQP3 (kfAQP3a) was cloned from the killifish gill, and a second homolog was identified as the consensus from a transcriptome database (kfAQP3b). The two were 99% homologous to each other, 98% homologous to a previously identified killifish AQP3 from embryos (kfAQP3ts), and 78% homologous to hAQP3. Expression of kfAQP3a in Xenopus oocytes significantly enhanced water, glycerol, and urea transport. However, kfAQP3a expressed in HEK293T cells did not transport significant amounts of arsenic. All sequence motifs thought to confer the ability of AQP3 to transport solutes were conserved in kfAQP3a, kfAQP3b, and kfAQP3ts; however, the C-terminal amino acids were different in kfAQP3a versus the other two homologs. Replacement of the three C-terminal amino acids of kfAQP3 (GKS) with the three C-terminal amino acids of kfAQP3b and kfAQP3ts (ANC) was sufficient to enable kfAQP3a to robustly transport arsenic. Thus, the C-terminus of kfAQP3b and kfAQP3ts confers arsenic selectivity in kfAQP3. Moreover, kfAQP3a, the only AQP expressed in killifish gill, is the first aquaglyceroporin identified that does not transport arsenic, which may explain, in part, why killifish poorly assimilate arsenic and are highly tolerant to environmental arsenic.

SUBMITTER: Jung D

PROVIDER: S-EPMC3327866 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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A novel aquaporin 3 in killifish (Fundulus heteroclitus) is not an arsenic channel.

Jung Dawoon D MacIver Bryce B Jackson Brian P BP Barnaby Roxanna R Sato J Denry JD Zeidel Mark L ML Shaw Joseph R JR Stanton Bruce A BA

Toxicological sciences : an official journal of the Society of Toxicology 20120208 1

The Atlantic killifish (Fundulus heteroclitus) is a model environmental organism that has an extremely low assimilation rate of environmental arsenic. As a first step in elucidating the mechanism behind this phenomenon, we used quantitative real-time PCR to identify aquaglyceroporins (AQPs), which are arsenite transporters, in the killifish gill. A novel homolog killifish AQP3 (kfAQP3a) was cloned from the killifish gill, and a second homolog was identified as the consensus from a transcriptome ...[more]

PMID: 22323512

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Project description:Arsenic is a contaminant found worldwide in drinking water and food. Epidemiological studies have correlated arsenic exposure with reduced weight gain and improper muscular development, while in vitro studies show that arsenic exposure impairs myogenic differentiation. The purpose of this study was to use Fundulus heteroclitus or killifish as a model organism to determine if embryonic-only arsenic exposure permanently reduces the number or function of muscle satellite cells. Killifish embryos were exposed to 0, 50, 200, or 800 ppb arsenite (AsIII) until hatching, and then juvenile fish were raised in clean water. At 28, 40, and 52 weeks after hatching, skeletal muscle injuries were induced by injecting cardiotoxin into the trunk of the fish just posterior to the dorsal fin. Muscle sections were collected at 0, 3 and 10 days post-injury. Collagen levels were used to assess muscle tissue damage and recovery, while levels of proliferating cell nuclear antigen (PCNA) and myogenin were quantified to compare proliferating cells and newly formed myoblasts. At 28 weeks of age, baseline collagen levels were 105% and 112% greater in 200 and 800 ppb groups, respectively, and at 52 weeks of age, were 58% higher than controls in the 200 ppb fish. After cardiotoxin injury, collagen levels tend to increase to a greater extent and take longer to resolve in the arsenic exposed fish. The number of baseline PCNA(+) cells were 48-216% greater in 800 ppb exposed fish compared to controls, depending on the week examined. However, following cardiotoxin injury, PCNA is reduced at 28 weeks in 200 and 800 ppb fish at day 3 during the recovery period. By 52 weeks, there are significant reductions in PCNA in all exposure groups at day 3 of the recovery period. Based on these results, embryonic arsenic exposure increases baseline collagen levels and PCNA(+) cells in skeletal muscle. However, when these fish are challenged with a muscle injury, the proliferation and differentiation of satellite cells into myogenic precursors is impaired and instead, the fish appear to be favoring a fibrotic resolution to the injury.

Project description:The Atlantic killifish (Fundulus heteroclitus), native to estuarine areas of the Atlantic coast of the United States, has become a valuable ecotoxicological model as a result of its ability to acclimate to rapid environmental changes and adapt to polluted habitats. MicroRNAs (miRNAs) are highly conserved small RNAs that regulate gene expression and play critical roles in stress responses in a variety of organisms. Global miRNA expression in killifish and the potential roles miRNA have in environmental acclimation have yet to be characterized. Accordingly, we profiled miRNA expression in killifish gill for the first time and identified a small group of highly expressed, well-conserved miRNAs as well as 16 novel miRNAs not yet identified in other organisms. Killifish respond to large fluctuations in salinity with rapid changes in gene expression and protein trafficking to maintain osmotic balance, followed by a secondary phase of gene and protein expression changes that enable remodeling of the gills. Arsenic, a major environmental toxicant, was previously shown to inhibit gene expression responses in killifish gill, as well the ability of killifish to acclimate to a rapid increase in salinity. Thus, we examined the individual and combined effects of salinity and arsenic on miRNA expression in killifish gill. Using small RNA sequencing, we identified 270 miRNAs expressed in killifish, and found that miR-135b was differentially expressed in response to arsenic and at 24?h following transfer to salt water. Predicted targets of miR-135b are involved in ion transport, cell motility and migration, GTPase mediated signal transduction and organelle assembly. Consistent with previous studies of these two environmental stressors, we found a significant interaction (i.e., arsenic dependent salinity effect), whereby killifish exposed to arsenic exhibited an opposite response in miR-135b expression at 24?h post hyperosmotic challenge compared to controls. By examining mRNA expression of predicted miRNA targets during salinity acclimation and arsenic exposure, we found that miR-135b targets were significantly more likely to decrease during salinity acclimation than non-targets. Our identification of a significant interaction effect of arsenic and salinity on miR-135b expression supports the hypothesis that arsenic alters upstream regulators of stress response networks, which may adversely affect the killifish response to osmotic stress. The characterization of miRNAs in this ecotoxicological model will be a valuable resource for future studies investigating the role of miRNAs in response to environmental stress.

Dataset Information

A novel aquaporin 3 in killifish (Fundulus heteroclitus) is not an arsenic channel.

Publications

A novel aquaporin 3 in killifish (Fundulus heteroclitus) is not an arsenic channel.

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