Abnormal nuclear morphology is independent of longevity in a zmpste24-deficient fish model of Hutchinson-Gilford progeria syndrome (HGPS).

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.

Responses of the CYP1A biomarker in Jenynsia multidentata and Phalloceros caudimaculatus and evaluation of a CYP1A refractory phenotype.

The level of cytochrome P450 1A (CYP1A) in fish is used as a typical environmental biomarker for the presence of organic contaminants. We used RT-qPCR to investigate CYP1A mRNA levels in the liver, gill and gonopodium of guppies Jenynsia multidentata and Phalloceros caudimaculatus in wetlands within the Rio Grande city (RG) which is under the influence of the Patos Lagoon Estuary (RS, Brazil). The CYP1A mRNA levels evaluated in fish liver from two locations that receive non-treated wastewater effluents (S3 and S4) and another locations near an oil refinery (S6) and an industrial complex (S7), were higher than in locations remote from those sites (S1, S2 and S5). The sum of 16 priority PAHs in sediment confirmed high levels in S4 and S6 (3914.0 and 4414.0 ng g(-1) dw, respectively) comparing to S7>S2>S3>S5>S1 (119.3, 66.3, 62.8, 16.4 and 1.7 ng g(-1) dw). J. multidentata from sites S1 to S4 that were transferred to the laboratory exhibited CYP1A induction after 24 h waterborne exposure to 1 µM betanaphtoflavone (BNF) in all organs compared to controls, except in the liver of fish from site S4. This lack of CYP1A induction by BNF indicates a CYP1A refractory phenotype in guppy. Although this characteristic possibly involves the alteration in AHR signaling or control, the mechanism of resistance is unknown. The present study provides information about the use of the use of CYP1A in South American guppies as an useful biomarker tool for environmental contamination studies.

Beta-naphthoflavone-inducedCYP1A expression in the guppy Jenynsia multidentata: Time-dependent response, anesthetic MS-222 effect and fin analysis.

Cytochrome P450 1A (CYP1A) expression in fish is used as a biomarker of exposure to organic contaminants, such PAHs, PCBs and dioxins, in the aquatic environment. South American guppy fish Jenynsia multidentata were exposed to the prototypical aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF; 1µM) and the fins were biopsied to characterize different aspects of CYP1A induction. RTq-PCR was used to quantify CYP1A mRNA levels in fish tissues. CYP1A induction in the gill, liver and anal fin (gonopodium) occurred within the first hour of waterborne exposure to BNF and persisted throughout 2, 4, 8, 24, 48 and 96h compared to controls (DMSO vehicle; p<0.05). The organ-specific temporal pattern of induction was marked by mRNA levels consistently augment as duration of exposure increases and tend to a sustained induction from 24h to 96h for gill and liver (∼15-fold and ∼50-fold over control, respectively). In gonopodium, there was a maximum CYP1A mRNA level at 4h (∼34-fold over control). Basal CYP1A mRNA levels and its induction following BNF exposure were not affected by administration of a chemical anesthetic (fish immersion in 100mgl(-1) MS-222 for 2-5min) in the gill, liver, gonopodium, dorsal or tail fin (p<0.05). In an ex vivo assay, in which small pieces of biopsied fins were exposed to BNF for 4h, high CYP1A induction was observed in the tail and gonopodium (∼49-fold and ∼69-fold, respectively) but not in the dorsal fin compared to controls. To our knowledge, this is the first study to show that a 1h waterborne exposure to an AHR agonist is sufficient to cause CYP1A induction in fish organs and fins. The present study added new information to the field regarding the use of MS-222 as an anesthetic on fish and the analysis of biopsied fins as an alternative non-lethalex vivo assay for evaluating the CYP1A biomarker in fish. This observation could be useful for planning fish toxicological bioassays and biomonitoring studies on the aquatic environments in South America.

Application of LDH-release assay to cellular-level evaluation of the toxic potential of harmful algal species.

Lactate dehydrogenase (LDH)-release assay was applied to estimate the toxic potential of harmful algal species at the cellular level. African green monkey kidney (Vero), yellowtail fin epithelia (MJF), and rainbow trout gill (RTgill-W1) cells were used as target cells. A live cell suspension of Karenia mikimotoi (SUO-1) induced the release of LDH from these cell lines, while the activity of another strain, FUK, was much lower. The cell-free culture supernatants and ruptured cell suspensions of both strains of K. mikimotoi were less effective on LDH-release assay. Exposure experiments against abalone and shrimp revealed that SUO-1 showed much stronger lethal effects on these organisms than FUK. Among six phytoplankton species, three species known to be harmful algal species induced the release of LDH to different extents depending on the cell line, whereas the other three species, known to be non-toxic, showed no effects on any cell lines. These results suggest that LDH-release assay is a useful micro-plate assay for estimation of the toxic potential of harmful phytoplankton.