Superfund Research Program
Aquatic Biomarkers in Site Characterization and Remediation
Project Leader: David E. Hinton (Duke University)
Grant Number: P42ES004699
Funding Period: 1995 - 2005
A tiny freshwater bony fish, the medaka, has proved to be an excellent experimental animal model for vertebrate carcinogenesis, mutagenesis, and genetics. Recently the see-through stock of medaka, genetically deficient in all four pigments, was developed and internal organs of living individuals may be seen through the transparent body wall. The researchers’ in vivo observations in intact individual see-through medaka have spanned embryonic, larval, and adult life stages. This characteristic is a significant advance because it overcomes the problem of pigment interference that precludes brightfield, conventional or widefield- and confocal- fluorescence imaging.
In medaka embryos, fluid contents of developing gall bladder are clear but soon become yellow in color, an indication that the individual has attained the capacity for bile formation. Observations over 25 days post fertilization (DPF) have included changes with onset of exogenous feeding, formation of a patent anus, and the ability to excrete bile into the gut and form feces. Because bile formation and excretion are critical for life, the project investigators’ in vivo studies have centered on hepato-biliary structure and function using bright field transmitted light and conventional fluorescence microscopy. After chemical induction of the liver enzyme, cytochrome P450 1A (CYP1A), liver cells of living embryo and larval fish were shown to cleave the substrate, ethoxyresorufin, and to transport the fluorescent product into the bile passageways. Without prior induction, this activity was not demonstrated. With the constitutive cytochrome P450 3A, cleavage of benzyloxyresorufin with subsequent biliary transport of the fluorescent product was demonstrated. This advance permits researchers to image critical life processes in vivo without harm to the individual. Using quantitative software packages and confocal microscopy, researchers now have a way to certify that early life stage exposure has led to toxic response and are able to follow the process through later life stages by in vivo systemic non-invasive analysis. In this way, the adult consequences of early life stage exposures may be discerned.
In separate studies with medaka, the scientists exploited suppression subtractive hybridization as a screening tool to assess qualitative gene expression changes in brain, liver, and testis and to identify 335 unique genes. Histological survey showed alterations in liver and testis but not brain. Based on real time polymerase chain reaction findings, relative CYP 1A (but not AHR1) transcript levels were confirmed to be significantly higher in dioxin (TCDD) exposed brain and liver but not in exposed testis. Gene expression and histopathology of testis may be independent of CYP1A. Unique liver and testis specific mRNA level targets in male medaka were identified as promising biomarkers of acute TCDD-induced toxicity. The significance of these findings is related to the team’s ability to anchor the morphologic response (phenotype) to the genotype.