A Pod of Sperm Whales Photo courtesy of Chris Johnson
Sperm whales are an endangered species (1). They are top predators in the marine food chain feeding primarily on squid and fish (2), much as humans are the top predator in their food chain. Sperm whales inhabit all oceans and they have a long life span (2). Thus they are an ideal ‘canary in the ocean’ for studying the threat of marine pollution.
In 2000, the research vessel Odyssey set sail from California embarking on a 5 year voyage circumnavigating the globe seeking sperm whales from around the world. The goal of the voyage was simple: Try to collect a global data set of ocean pollution using the sperm whale as an indicator species in an effort to see how badly polluted our oceans are. In 2004, we met Ocean Alliance and joined this effort as a close partner. Their goal is to measure levels in sperm whales from around the world and ours is to extend those data and determine how toxic these chemicals are to whale cells and how the effects on whale cells compare to human cells. The voyage is now complete and the lab work has begun! Some exciting results from this unprecedented effort are below.
We measured the levels of several essential and non-essential elements (total of 22: aluminium (Al), arsenic (As), barium (Ba), berelium (Be), boron (B), cadmium (Cd), chromium (Cr), copper (Co), iron (Fe), mercury (Hg), magnesium (Mn), molybdenum (Mo), niquel (Ni), potassium (K), sodium (Na), silver (Ag), sulfur (S), titanium (Ti) and zinc (Zn) in sperm whale tissue collected during the Voyage of the Odyssey.
The analysis showed that various environmental contaminants were present in sperm whale skin tissue.
Interestingly, we found levels of the heavy metal chromium, a well known human carcinogen, in relatively high levels in sperm whale skin tissue.
|This graphs shows chromium levels in sperm whale skin tissues.|
We are also investigating the effects of these contaminants on cell death, and chromosome and DNA damage in cell lines developed from sperm whale skin. With the data obtained from these efforts, we will be able to determine:
| Sperm Whale Skin Cells
(10X microscope magnification)
1) Which classes of pollutants are elevated around the world;
2) Which pollutants pose the largest risk to whales;
3) Which pollutants the whales respond to in a manner similar to humans; and
4) Where are the most polluted areas around the world.
This study will greatly enhance our knowledge of the physiology and toxicology of the sperm whale. Moreover, it will create tools (cell lines) that can serve as sperm whale-specific models, which can be used by other investigators to better understand additional aspects of sperm whale genetics, physiology, immunology and biochemistry, as well as investigations into the effects of other contaminants and infectious agents.
Chromosomal instability is a hallmark of cancer, and refers to abnormal alterations in chromosomes. These alterations can be in form of changes in the number of chromosomes (due to loss or gains of chromosomes) and/or aberrations in the structures of the chromosomes.
To study chromosomal aberrations we analyze metaphases from sperm whale’s treated cells. The most common chromosomal aberrations are chromatid lesions (breaks in one arm of the chromosome, where the width of the break is wider that the width of the chromatid) and gaps (breaks in one arm of the chromosome, where the width of the break is narrower that the width of the chromatid), and isochromatid lesions and gaps (breaks in both arms of the chromosomes).
|Examples of damaged chromosomes in sperm whale (100X microscope magnification):
Chromatid lesions (red arrow) and chromatid gap (green arrow).
We investigated the toxic effects of hexavalent chromium, a well know human carcinogen in the sperm whale skin cells.
Chromium caused cell death in sperm whale skin cells.
|This graph shows that soluble chromium (VI) is cytotoxic to sperm whale skin cells.|
Chromium also caused DNA damage (measured as amount of chromosomal aberrations) in all cell types, as well as, cell cycle arrest (cell’s inability to divide), as represented by NM (=no metaphases).
|This graph shows that soluble chromium (VI) induces chromosome damage in sperm whale skin cells.|
We are further investigating the potential harmful effects of trivalent chromium [Cr(III)], that some believe to be an essential element. However, a few studies on mammalian cells have shown that Cr(III) can also induce chromosome damage. Visit us soon for more updates!
All cells, being mammalian or non-mammalian, have conserved protection mechanisms against DNA damage. These cellular mechanisms can be specific to the different types of DNA damage.
DNA double strand breaks is one type of DNA damage that have been observed to be repaired. We perform two different assays to investigate this type of DNA damage:
- The single cell gel electrophoresis (Comet assay)
In this assay several single cell nuclei are observed on a gel-coated microscope slide. In a nucleus with DNA double strand breaks, these pieces of DNA double strands (negatively charged) will move towards the positive end of the electrophoresis unit generating a “tail”, which will then resemble a comet:
|Sperm whale cell nuclei with no DNA double strand breaks.|
|Sperm whale cell nucleus with DNA double strand breaks (nuclei with "tails").|
- H2A.X immunofluorescence
In this assay, DNA double strand breaks are detected indirectly by analyzing a protein that is known to give the signal to the cell of the presence of the break, the H2A.X. Upon the formation of a DNA double strand breaks, several H2A.X proteins in the vicinity of the break are activated forming a focus of activated proteins. This focus can be visualized by immunofluorescence methods, where each focus (green dot) is known to represent one single DNA double strand break (the nucleus is stained in blue):
|This picture shows sperm whale skin nuclei with and without DNA damage, in forms of DNA double strand breaks.
One green focus corresponds to one DNA double strand break.
Repair of this type DNA damage can be done by observing whether “tails” or with “green dots” disappear over time, in damaged cells. Soon we will be posting exciting data on DNA damage repair in the sperm whale cells!
Cells that we develop from whale biopsies are primary cells which have a limited life-span. And therefore, we are trying to immortalizing sperm whale cells by inserting a human telomerase reverse transcriptase gene (hTERT) using the green-fluorescent protein (GFP)-tagged gene technique. hTERT is expressed in a small group of cells, including stem cells and cancer cells, and is the catalytic component of the enzyme telomerase
Most of cells have a limited life-span, they can only divide for a limited number of times, and these is due to the shortening of the chromosomes each time the cell divides. However, cells do not loose important genetic information, because they have segments of repetitive DNA at the end of the chromosomes called telomeres. But when the telomeres are used up, the cell senesces.
Telomeres can be maintained by the enzyme telomerase, and thus preventing the cell to senesce. And this is why we are trying to insert the hTERT gene into sperm whale cells.
The GFP-tagged gene technique is a technique that enables us to confirm the insertion of a given gene by only looking at the cells under a microscope. Green cells tell us that the gene was successfully inserted.
|Sperm whale skin cells infected with GFP-hTERT (under normal light).|
|Sperm whale skin cells infected with GFP-hTERT (under fluorescent light).|
(soon to come!)
1. Carretta, J., Forney, K.A., Muto, M.M., Barlow, J., Baker, J., Hanson, B., and Lowry, M.S. U.S. Pacific Marine Mammal Stock Assessments: 2004. NOAA Technical Memorandum NMFS 1-322, 2005.
2. Marine Fisheries Review. The Sperm Whale. 61(1):59-74, 1999.
Relevant Wise Laboratory Publications
Li Chen, T., LaCerte, C., Wise, S.S., Holmes, A., Martino, J., Wise, Jr., J.P., Thompson, W.D. and Wise, Sr., J.P. Comparative Cytotoxicity and Genotoxicity of Particulate and Soluble Hexavalent Chromium in Human and Sperm Whale (Physeter macrocephalus) Skin Cells. Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology, 155: 143–150, 2012. (PMID: 21466859)
Wise, Sr., J.P., Thompson, W.D., Wise, S.S., LaCerte, C., Wise, J., Gianios, Jr., C., Perkins, C., Zheng, T., Benedict, L., Mason, M., Payne, R. and Kerr, I. A Global Assessment of Gold, Titanium, Strontium and Barium Pollution using Sperm Whales (Physeter macrocephalus
Wise, Sr., J.P., Wise, S.S., LaCerte, C., Wise, Jr., J.P. and Aboueissa, A-H., The Genotoxicity of Particulate and Soluble Chromate in Sperm Whale (Physeter macrocephalus) Skin Fibroblasts by Environmental and Molecular Mutagenesis, Environmental and Molecular Mutagenesis, 52: 43–49, 2011.
Wise, Sr., J.P., Payne, R., Wise, S.S., LaCerte, C., Wise, J., Gianios, Jr., C., Thompson, W.D., Perkins, C., Zheng, T., Zhu, C., Benedict, L. and Kerr, I. A Global Assessment of Chromium Pollution using Sperm Whales (Physeter macrocephalus) as an Indicator Species. Chemosphere.75: 1461–1467, 2009.
Collaborators and Cooperators
The Wise Laboratory is assisted in this work by an important number of collaborators and cooperators. In particular, the following prominent scientists and their teams provide significant support and input:
Dr. AbouEl-Makarim Aboueissa is an Assistant Professor of Mathematics and Statistics at the University of Southern Maine (USM). He provides statistical expertise particularly in the area of handling samples with measurements at or below detection limits.
Mr. Iain Kerr is the Vice President and Chief Executive Officer of The Ocean Alliance. He provides access to the research vessel Odyssey and access to whale samples. Dr. Roger Payne is the Founder and President of The Ocean Alliance in Lincoln, Massachusetts. He provides expert advice and guidance on identification and geographic distribution of whales and other marine mammals. Dr. Yawei Zhang is an Assistant Professor of Epidemiology and Public Health at Yale University. She provides expert advice and guidance on the statistical analysis and epidemiological design of marine mammal studies. Dr. Tongzhang Zheng is Professor of Epidemiology and Public Health and Head of the Environmental Health Sciences Division at Yale University. He provides expert advice and guidance on the statistical analysis and epidemiological design of marine mammal studies. Funding This work is generously supported by the Ocean Alliance and the Maine Center for Toxicology and Environmental Health.
Mr. Iain Kerr is the Vice President and Chief Executive Officer of The Ocean Alliance. He provides access to the research vessel Odyssey and access to whale samples.
Dr. Roger Payne is the Founder and President of The Ocean Alliance in Lincoln, Massachusetts. He provides expert advice and guidance on identification and geographic distribution of whales and other marine mammals.
Dr. Yawei Zhang is an Assistant Professor of Epidemiology and Public Health at Yale University. She provides expert advice and guidance on the statistical analysis and epidemiological design of marine mammal studies.
Dr. Tongzhang Zheng is Professor of Epidemiology and Public Health and Head of the Environmental Health Sciences Division at Yale University. He provides expert advice and guidance on the statistical analysis and epidemiological design of marine mammal studies.
This work is generously supported by the Ocean Alliance and the Maine Center for Toxicology and Environmental Health.