Hypoxia means that oxygen levels are depleted, which threatens aquatic species. Coastal marine waters provide an essential economic and ecological resource worldwide, but are increasingly threatened by pollution and development. Our study focuses on the Gulf of Mexico, where heavy metal and petroleum pollution pose serious ecological threats, and hypoxia is believed to be causing a 3,000 square mile "dead zone" in which marine life is virtually absent.
Chromium, primarily in its hexavalent form [Cr(VI)], is a common heavy metal pollutant in marine waters. It is often found at high levels near metal finishing industries, municipal treatment plants, oil drilling operations and cooling towers (1). Cr(VI) has been shown to be mutagenic, carcinogenic and teratogenic in a variety of organisms. Its effect on fish cells is yet unknown.
Hypoxia not only causes mass mortality events, but also has significant physiological effects on marine organisms (2). The goal of this project is to investigate how chromium affects fish cells, and whether hypoxic conditions alter these effects. Our research organism is medaka, a small, freshwater teleost fish that is a well-established model organism for toxicological, genetic and developmental research. All experiments will be performed with OLHNI2 cells derived from the fin of HNI strain adult medaka. Additionally, we will attempt to establish cell lines from a lacl transgenic medaka with the goal of conducting mutagenesis assays in the future.
|Figure 1: This figure shows (A) OLHNI2 cells in culture and (B) a normal metaphase in OLHNI2 cells. These cells have 48 chromosomes.
We have characterized medaka cells (OLHNI2) in culture for use with standard cytotoxicity and genotoxicity assays. We found that sodium chromate is cytotoxic to medaka cells, causing little survival after exposure with 100uM for 24 h (Figure 2). We measured DNA double-strand breaks using an immunofluoresence assay to visualize gamma-H2A.X foci, which form at DNA double-strand break sites (Figure 3). We also investigated the clastogenicity of sodium chromate by analyzing chromosome damage in normal metaphase spreads. We found that sodium chromate caused DNA double-strand breaks and chromosome damage in medaka cells, which increased with concentration. The effects of hypoxia on fish cells will be studied using chambers that can be filled with different gas mixtures and sealed. We will also determine the uptake of sodium chromate by measuring chromium ion concentration with inductively coupled plasma atomic emission spectrometry (ICP-AES).
|Figure 2. Treatment with sodium chromate causes a concentration-dependent decrease in survival in OLHNI2 cells at both the 24 and 120 h timepoints.|
Figure 4. Sodium chromate treatment causes a concentration-dependent increase in the number of gamma-H2A.X foci per cell.
Figure 5. Cells are treated with different oxygen mixtures in sealed chambers.
Figure 6. This figure shows a metaphase with chromatid exchanges, a form of chromosome damage.
- Sadiq, Muhammad. "Chromium in Marine Environments" in: Toxic Metal Chemistry in Marine Environments, Marcel Dekker Inc. New York, New York. 1992.
- Wu, Rudolf S.S. Hypoxia: from molecular responses to ecosystem responses. Marine Pollution Bulletin 45: 35-45, 2002.
Goodale, B.C., Walter, R., Pelsue, S.R., Thompson, W.D., Wise, S.S., Winn, R.N., Mitani, H., Wise Sr., J.P. The Cytotoxicity and Genotoxicity of Hexavalent Chromium in Medaka (Oryzias latipes) Cells. Aquatic Toxicology 87: 60–67, 2008.
The Wise Laboratory is assisted in this work by Dr. Ronald Walter, who is Professor of Chemistry and Biochemistry, and Director of the Xiphophorous Genetic Stock Center at Texas State University at San Marcos. He provides expertise and direction on the proteomics and genomics aspects of the project.
This work is generously supported by grant number NA04NOS4260202 from the US Department of Commerce, National Oceanic and Atmospheric Administration, the Maine NSF EPSCOR Forest Bioproducts Research Project and by the Maine Center for Toxicology and Environmental Health.