Japanese Medaka Embryos Research Articles

NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis

Fish embryo toxicity tests for chemical risk assessment have traditionally been based upon non-specific endpoints including morphological abnormalities, hatching success, and mortality. Here we extend the application of 1H NMR-based metabolomics in environmental toxicology by adding a suite of metabolic endpoints to the Japanese medaka (Oryzias latipes) embryo assay, with the goal to provide more sensitive, specific and unbiased biomarkers of toxicity. Medaka were exposed throughout embryogenesis to five concentrations of trichloroethylene (TCE; 0, 8.76, 21.9, 43.8, 87.6, 175 mg/L) and the relative sensitivities of the traditional and metabolomic endpoints compared. While the no-observable-adverse-effect-level for hatching success, the most sensitive traditional indicator, was 164 mg/L TCE, metabolic perturbations were detected at all exposure concentrations. Principal components analysis (PCA) highlighted a dose-response relationship between the NMR spectra of medaka extracts. In addition, 12 metabolites that exhibited highly significant dose-response relationships were identified, which indicated an energetic cost to TCE exposure. Next, embryos were exposed to 0, 0.88, 8.76 mg/L TCE and sampled on each of the 8 days of development. Projections of 66 two-dimensional J-resolved NMR spectra were obtained, and PCA revealed developmental metabolic trajectories that characterized the basal and TCE-perturbed changes in the entire NMR-visible metabolome throughout embryogenesis. Although no significant increases in mortality, gross deformity or developmental retardation were observed relative to the control group, TCE-induced metabolic perturbations were observed on day 8. In conclusion, these results support the continued development of NMR-based metabolomics as a rapid and reproducible tool for biomarker discovery and environmental risk assessment.

The interaction effects of binary mixtures of benzene and toluene on the developing heart of medaka ( Oryzias latipes)

The United States Environmental Protection Agency (USEPA) has pursued the estimation of risk of adverse health effects from exposure to chemical mixtures since the early 1980s. Methods used to calculate risk estimates of mixtures were often based on single chemical information that required assumptions of dose-addition or response-addition and did not consider possible changes in response due to interaction effects among chemicals. Full factorial designs for laboratory studies can produce interactions information, but these are expensive to perform and may not provide the information needed to evaluate specific environmentally relevant mixtures. In this research, groups of Japanese medaka ( Oryzias latipes) embryos were exposed to binary mixtures of benzene and toluene as well as to each of these chemicals alone. Endpoint specific dose–response models were built for the hydrocarbon mixture under an assumption of dose-additivity, using the single chemical dose–response information on benzene and toluene. The endpoints included heart rate, heart rate progression, and lethality. Results included a synergistic response for heart rate at 72 h of development, and either additivity or antagonism for all other endpoints at 96 h of development. This work uses an established statistical method to evaluate the toxicity of an environmentally relevant mixture to ascertain whether interaction effects are occurring, thus providing additional information on toxicity.

Effects of bis(2-ethylhexyl) phthalate and benzo[a]pyrene on the embryos of Japanese medaka ( Oryzias latipes)

Effects of two widely found chemical pollutants, bis(2-ethylhexyl) phthalate (DEHP) and benzo[a]pyrene (BaP), on the embryos of Japanese medaka were investigated. The embryos were exposed to different concentrations (0.01, 0.1, 1, and 10 μg/l) of DEHP and BaP. The following were investigated: (1) hatching time and hatching rate in embryos, (2) mortality, sex ratio, body weight and gonadosomatic index (GSI) in adulthood. These two chemicals delayed the hatching time without dose-dependence, but these chemicals had no effect on hatching rate. Mortality was raised and body weight was reduced by DEHP and BaP-treatment; distortion of sex ratio appeared at the lowest concentration of DEHP tested. GSI was decreased because of the BaP-treatment. DEHP and BaP negatively affected Japanease medaka embryos, and the influences of the effects continued into adult stage. Moreover, the effects did not appear to be necessarily dose-dependent.

Immunocytochemical localization of epidermal growth factor receptor in early embryos of the Japanese medaka fish (Oryzias latipes).

This study was undertaken to localize epidermal growth factor receptor (EGFR) during early development of Japanese medaka embryos using immunocytochemistry. Specific staining was observed in all stages studied. All of the cells of the embryonic disc from the germinal disc (1 cell) through the late high blastula stages stained moderately for EGFR. Beginning with the flat blastula stage, the surface and lateral cells of the embryonic disc and the cells migrating around the yolk stained intensely for EGFR, and this continued throughout the study period. The presence of the keel at the late gastrula stage did not affect the moderate staining of the majority of the embryonic disc cells. When somites first appeared, the keel region stained less intensely than before, but scattered individual cells stained intensely for EGFR. Embryos with 12 somites had a neural tube that was lightly stained except for a few intensely stained individual cells. The neural tube, notochord and somites in 24-somite embryos lacked immunostaining. However, the surface epithelium, aorta, intestinal epithelium and pronephric duct demonstrated EGFR immunostaining. This study demonstrates that EGFR is present during medaka development and supports the hypothesis that EGFR ligands are important during cleavage, gastrulation and early organogenesis.

Effects of Acrobat MZ® and Tattoo C® on Japanese medaka ( Oryzias latipes) development and adult male behavior

Exposure of Japanese medaka ( Oryzias latipes) embryos and adult males to sublethal concentrations of two commonly used fungicides, Acrobat MZ and Tattoo C, had varying effects on development and adult male behavior. During embryolarval assays, medaka exposed to Acrobat exhibited decreased heart rates throughout embryonic development and were smaller at hatching than controls. Differences in hatching size were not apparent with Tattoo although some decrease in heart rate was observed at higher concentrations. Tail lesions and abnormal spinal development was a problem common to embryos exposed to Acrobat. Neither time to hatch nor early growth was affected by exposure to either fungicide. Adult males that had been exposed to high concentrations of Acrobat or Tattoo over a 28-day period were less likely to approach females during behavioral trials. The absence of other behavioral effects, particularly those related to reproductive performance, may have been due to females being sexually unreceptive during behavioral trials. No other behavioral modifications were apparent for adult males exposed to either fungicide. Information about developmental and behavioral responses to sublethal concentrations of pesticides is important for establishing environmental guidelines concerning their use.

Modifications of the Topical Japanese Medaka (Oryzias latipes) Embryo Larval Assay for Assessing Developmental Toxicity of Pentachlorophenol and p, p′-Dichlorodiphenyltrichloroethane

One method currently available for investigating developmental toxicity in teleost species is the Japanese medaka embryo larval assay (MELA). In the present study, the MELA was modified to evaluate repeated topical exposures to pentachlorophenol (PCP) and p, p′-dichlorodiphenyltrichloroethane (DDT) and to identify sensitive stages of embryonic development. A single topical exposure using embryos at 48 h postfertilization resulted in a statistically significant increase in embryo mortality at 688 and 1250 ng PCP/egg compared with controls. In contrast, the toxicity following exposure to 11, 36, 78, 120, 208, and 400 ng DDT/egg was expressed only in larvae after hatching. Results further demonstrate that the MELA can be optimized to accommodate repeated daily topical exposures starting at 48 h postfertilization and ending at 120 h postfertilization. In addition, the neurula stage (24 h postfertilization) represented the most sensitive embryonic stage following a single topical exposure of PCP. However, no differences were observed in the sensitivity of embryonic stages following DDT exposure. The modified MELA was also used to evaluate sediment extracts contaminated with DDT metabolites obtained from the Tensas River, Louisiana. Results indicate that there is a low potential for developmental toxicity using the present extraction and exposure scenario even though elevated levels of DDE and toxaphene currently exist in several adult fish species at this site. The MELA as a screen for evaluating the potential for developmental toxicity of contaminated sediments is discussed.

Interactions of 2,3,7,8-tetrachlorodibenzo- P-dioxin (TCDD) and 3,3′4,4′5-pentachlorobihenyl (PCB 126) for producing lethal and sublethal effects in the japanese medaka embryos and larvae.

Newly fertilized eggs and one-month-old larvae of Japanese medaka ( Oryzias latipes) were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), or their mixtures to evaluate their interaction using several toxic end points. The uptake of TCDD and PCB 126 in a binary mixture increased with dose and was not significantly different from that of each congener alone. The elimination of TCDD and PCB 126 in the binary mixture in the Japanese medaka larvae was also not significantly different from that of the individual congener. PCB 126 (94 – 300 pg/ml) and TCDD (1.8 - 6.2 pg/ml) acted additively to produce mortality and the other toxic effects measured in the Japanese medaka embryos and larvae at the average ratios of 47:1 and 55:1, respectively.

Sex on the Brain but the Heart Is Not Really In It: Developmental Heart Defects Associated with Aquatic Pollution and Microinjection of Hexachlorobenzene into the Japanese Medaka Embryo.

Previous articleNext article No AccessDEVELOPMENTSex on the Brain but the Heart Is Not Really In It: Developmental Heart Defects Associated with Aquatic Pollution and Microinjection of Hexachlorobenzene into the Japanese Medaka EmbryoM. Mizell, E. Romig, W. Hartley, and A. ThiyagarajahM. Mizell Search for more articles by this author , E. Romig Search for more articles by this author , W. Hartley Search for more articles by this author , and A. Thiyagarajah Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 189, Number 2October 1995 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv189n2p196 Views: 11Total views on this site Citations: 2Citations are reported from Crossref Copyright © 1995 by Marine Biological LaboratoryPDF download Crossref reports the following articles citing this article:M. Mizell, E. Romig, J. Stegeman, R. Smolowitz, and R. Katayani Zebrafish Embryo Monitoring of the Aquatic Environment: Dose-Response Synergism Revealed in Combinations of Pollutant Chemical Mixtures, The Biological Bulletin 191, no.22 (Sep 2016): 292–294.https://doi.org/10.1086/BBLv191n2p292M. Mizell, J. J. Stegeman, E. Romig, R. Smolowitz, J. Schlezinger, R. Katayani, B. Woodin, and M. Mortensen Chemically Induced Cardiovascular Defects in Developmental Stages of Vertebrates: Dose-Response and Phenotypic Comparisons in Medaka and Zebrafish Exposed to Aryl Hydrocarbon Receptor Agonists, The Biological Bulletin 191, no.22 (Sep 2016): 294–295.https://doi.org/10.1086/BBLv191n2p294

Embryotoxicity of extracts from lake ontario rainbow trout (oncorhynchus mykiss) to japanese medaka (oryzias latipes)

AbstractVarious preparative techniques were used to extract nonpolar organic compounds from the muscle tissue of Lake Ontario rainbow trout (Oncorhynchus mykiss). In this extract, PCBs and organochlorine compounds were detected in nanogramper‐milliliter quantities, and polychlorinated dibenzo‐p‐dioxins and dibenzofurans were detected in picogram‐per‐milliliter quantities. The extract and various subfractions of the extract were tested for embryotoxicity in a bioassay with embryos of Japanese medaka (Oryzias latipes). The whole extract was embryotoxic to medaka, as were an extract fraction containing PCBs (fraction A) and extract fractions containing nonpolar organochlorine compounds (fractions B and C). When subfractions prepared from fraction A were tested for embryotoxicity, a subfraction containing non‐ortho‐substituted PCB congeners was embryo‐toxic, whereas subfractions containing mono‐ortho‐ and di‐ortho‐substituted PCB congeners were relatively nontoxic. Pathological lesions characteristic of exposure to planar halogenated aromatic hydrocarbons were observed only in embryos exposed to the non‐ortho‐PCB subfraction. The non‐ortho‐PCB subfraction of fraction A was more toxic than the original fraction A, which indicates that nontoxic PCBs reduce the toxicity of the non‐ortho‐PCBs through some unknown mechanism. This study indicates that organochlorine compounds and non‐ortho‐substituted PCBs have the potential to be embryotoxic to early life stages of Great Lakes fish, but nontoxic contaminants can modify this toxic response. These data are relevant to the interpretation of correlations between embryo mortalities and concentrations of persistent organic contaminants in Great Lakes salmonids.

EMBRYOTOXICITY OF EXTRACTS FROM LAKE ONTARIO RAINBOW TROUT (ONCORHYNCHUS MYKISS) TO JAPANESE MEDAKA (ORYZIAS LATIPES)

Various preparative techniques were used to extract nonpolar organic compounds from the muscle tissue of Lake Ontario rainbow trout (Oncorhynchus mykiss). In this extract, PCBs and organochlorine compounds were detected in nanogramper-milliliter quantities, and polychlorinated dibenzo-p-dioxins and dibenzofurans were detected in picogram-per-milliliter quantities. The extract and various subfractions of the extract were tested for embryotoxicity in a bioassay with embryos of Japanese medaka (Oryzias latipes). The whole extract was embryotoxic to medaka, as were an extract fraction containing PCBs (fraction A) and extract fractions containing nonpolar organochlorine compounds (fractions B and C). When subfractions prepared from fraction A were tested for embryotoxicity, a subfraction containing non-ortho-substituted PCB congeners was embryo-toxic, whereas subfractions containing mono-ortho- and di-ortho-substituted PCB congeners were relatively nontoxic. Pathological lesions characteristic of exposure to planar halogenated aromatic hydrocarbons were observed only in embryos exposed to the non-ortho-PCB subfraction. The non-ortho-PCB subfraction of fraction A was more toxic than the original fraction A, which indicates that nontoxic PCBs reduce the toxicity of the non-ortho-PCBs through some unknown mechanism. This study indicates that organochlorine compounds and non-ortho-substituted PCBs have the potential to be embryotoxic to early life stages of Great Lakes fish, but nontoxic contaminants can modify this toxic response. These data are relevant to the interpretation of correlations between embryo mortalities and concentrations of persistent organic contaminants in Great Lakes salmonids.

Fish-embryo toxicity associated with exposure to soils and sediments contaminated with varying concentrations of dioxins and furans

Studies were carried out to evaluate the toxicity to Japanese medaka ( Oryzias latipes) embryos after exposure to soils and sediments from various sites contaminated with dioxins and furans. Eggs were exposed to 1–10 mg of material in 1 ml of rearing solution held in a glass vial. The eggs were monitored on a daily basis for death, stage development, and lesions. The soils that were evaluated included: two sites from Newark, NJ (2200 and 180 μg/kg 2,3,7,8-TCDD); Times Beach, Missouri (950 μg/kg 2,3,7,8-TCDD); and Seveso, Italy (128 μg/kg 2,3,7,8-TCDD). Exposure to these soils resulted in 100% lethality. Based on the Newark, NJ, soils, the LC50 to post-hatch survival was 0·09 (0·01–0·93) μg/kg of TCDD soil-bound equivalents. Exposure to estuarine core sediments collected near the Newark, NJ, site that contained 0·73–21 μg/kg were also toxic to the embryo, but the toxicity was less in the sediments than in the soil. The estimated bioavailability from the Newark soil was less than 0·2%. This type of biological-based toxicity can be useful in evaluating the extent of clean-up that may be required for any specific site.

Evaluation of baltic herring and icelandic cod liver oil for embryo toxicity, using the japanese medaka (oryzias latipes) embryo larval assay

AbstractThese studies were carried out to determine if oils obtained from wild fish populations were contaminated with polychlorinated‐p‐dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) and, if they were contaminated, would they result in embryo toxicity as measured by the Japanese medaka (Oryzias latipes) embryo larval bioassay (ELA). Three fish oils [Baltic herring oil (HO 1); Baltic herring oil spiked with 44 parts per trillion (pptr) of 1,2,3,7,8,9‐HxCDD, 120 pptr OCDD and 130 pptr 2,3,7,8‐TCDF (HO 2*); and a commercially available cod liver oil (CL)] and a corn oil sample were tested for embryo toxicity in a static renewal protocol. The ELA assay was selected due to its sensitivity to biological effects following dioxin and dibenzofuran exposure. Two of the fish oils (HO 1 and HO 2*) were from Baltic herring collected from the Gulf of Bothnia, Sweden, and the third was a commercially available Icelandic cod liver oil. The Baltic herring oil contained higher levels of both PCDDs and PCDFs than the cod liver oil: 2,3,7,8‐TCDD, 5X; 1,2,3,7,8‐PeCDD, 5X; 1,2,3,6,7,8‐HxCDD, 6X; 2,3,7,8‐TCDF, 3X; 1,2,3,7,8,‐PeCDF, 3X; 2,3,4,7,8‐PeCDF, 4X; 1,2,3,4,7,8/1,2,3,4,7,9‐HxCDF, 3X; 1,2,3,6,7,8‐HxCDF, 4X; and 1,2,3,4,6,7,8‐HpCDF, 2X. The HO 1 sample resulted in 30 to 90% death, whereas the HO 2* sample caused 100% death. The major lesions were hemorrhage, cardiac effects and tissue necrosis. The time to lesion occurrence and the types of lesions were similar to those observed in studies using pure 2,3,7,8‐TCDD. The rearing solution control, corn oil control and cod liver oil samples caused no mortality or lesions. The lethality and lesions caused by these oils in the ELA system were similar to those reported in Salmo Salter with the M74‐syndrome from Sweden. The use of contaminated fish oils, therefore, may be one cause of poor viability in hatchery salmon reared in Sweden, and care should be taken when using naturally occurring fish or fish oils in nondefined feeds.

The stage specific toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in embryos of the japanese medaka (Oryzias latipes)

Embryos of the Japanese medaka were individually exposed to varying concentrations of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a static, nonrenewal system. The EC50 with 95% confidence interval (C.I.) to prevent hatching was 14 (11–17) nanograms (ng) of TCDD equivalents per liter (L) of water (parts per trillion). The LC50 with 95% C.I. for survival to 3 d posthatch was 9 (6–12) ng TCDD equivalents/L. The EC50 with 95% C.I. for embryos with minor lesions and severe, life-threatening lesions were 3.5 (1.3–5.7) ng TCDD equivalents/L and 14 (12.4–15.6) ng TCDD equivalents/L, respectively. In a separate experiment that was terminated prior to the embryos hatching or dying, the EC50 for lesions was calculated to be 2.2 (1.4–3.0) ng TCDD equivalents/L. Based on the amount of TCDD equivalents recovered from dechorionated embryos, the ED50 with 95% C.I. for lesions was calculated to be 0.24 picograms of TCDD equivalents per milligram of dechorionated embryo weight (parts per billion). When the embryos were exposed to [3H]TCDD within 1 to 2 h after fertilization, no concentration dependent increase in visible lesions was observed until after the formation of the liver rudiment (day 4 of development). By exposing Japanese medaka embryos to lethal concentrations of TCDD beginning on different days of embryonic development, it was demonstrated that the sensitive period for toxicity was during liver formation on day 4 or 5 of development. The sensitive period for development was not caused by differences in TCDD absorption across the chorion. When embryos were exposed to [3H]TCDD prior to, during or after liver formation, there was no statistical difference in the dose of TCDD equivalents that crossed the chorion and entered the yolk and embryo.

Age-dependent changes in toxicity of N-nitroso compounds to Japanese medaka ( Oryzias latipes) embryos

Three different developmental stages of embryonated eggs of Oryzias latipes were exposed to 2-h pulses of a number of different concentrations of MNU, MNNG, and DENA. Lethality and teratogenic endpoints were assessed through 24 h posthatch. MNU (≥2.5 mM) and MNNG (≥0.75 mM) exposure at the multicell stage was lethal, but the same exposure during early organogenesis was largely teratogenic. Four days before hatching, embryos were very resistant to MNU and proceeded to hatch normally; 7.5 mM MNU was required to significantly reduce the percent normal hatch. Effects of DENA were equivocal and only seen at the multicell stage. In order to differentiate between potential changes in chorion permeability, and toxicity per cell, the same three stages were exposed to a series of radiolabeled compounds of varying hydrophobicity, and apparent uptake measured. There were no differences in uptake rate or equilibrium concentration between any of the compounds at the different stages. These results suggest that, as in mammals, certain stages of development are more susceptible than others, and that these differences are a result of embryo sensitivity rather than chemical bioavailability. The most sensitive indicator of exposure was posthatch inflation of the swimbladder. Other terata included pericardial edema, anisophthalmia, and partial rupture of the chorion.

Comparison of the toxicity of several polychlorinated dibenzo-p-dioxins and 2,3,7,8-tetrachlorodibenzofuran in embryos of the Japanese medaka ( Oryzias latipes)

Nine polychlorinated dibenzo-p-dioxins (PCDDs) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were tested in the Japanese medaka embryo bioassay. Individual embryos were exposed to varying nominal concentrations of a PCDD or 2,3,7,8-TCDF under static conditions. The LC 50 with 95% confidence intervals for survival to 3 days post-hatch were as follows: 2,3,7,8-TCDD, 13 (10–17) nanograms (ng)/liter (L) (parts per trillion); 2,3,7,8-TCDF, 16 (12–22) ng/L; 1,2,3,7,8-PeCDD, 27 (23–31) ng/L and 1,2,3,4,7,8-HCDD, 2900 (718–11717) ng/L. Both 1,2,7,8-TCDD and 2,3,7-TriCDD caused a concentration dependent increase in visible lesions from 50 to 50000 ng/L. In the 1,2,7,8-TCDD exposed embryos, lethality was observed only at the highest concentration tested (50000 ng/L). For 2,3,7-TriCDD, there was no concentration related increase in lethality from 50 to 50000 ng/L. At 50000 ng/L, 1,3,6,8-TCDD, 2,8-DCDD and 2,3-DCDD did not cause any visible toxicity. The octachlorodibenzo-p-dioxin congener did not cause any visible toxicity at 10000 ng/L. The toxic PCDDs and 2,3,7,8-TCDF caused the same sequela of lesions in the developing Japanese medaka, suggesting that they may act through a common mechanism. These results demonstrate that 2,3,7,8-TCDF and PCDDs with the four lateral positions chlorine substituted and at least one other position non-substituted are toxic to the Japanese medaka embryo. The structure activity relationship observed here is similar to that observed in guinea pigs, mice and trout eleutheroembryos.