The North Atlantic right whale is one of the most severely endangered large whales, with only about 400 animals left in their population. The underlying causes for the inability of these animals to recover in population size are uncertain and are likely to be a combination of factors. A recent international conference of marine mammal experts concluded that "...right whales are routinely exposed to a wide array of xenobiotic chemicals, some of which generate toxic effects on mammalian reproductive and immune systems", and that "...significant fundamental research was needed to adequately assess the effects of pollutants on cetaceans". Thus one possible factor contributing to the reduced population is the effect of environmental chemicals on the reproductive tissues of North Atlantic right whales.
By contrast, the South Atlantic right whales are a healthy population with several thousand members. This difference in health strongly implicates an environmental factor in the decline of the North Atlantic right whale population. Each year we collect field samples from each population traveling to Lubec, Maine (North Atlantic right whales) and Patagonia in Argentina (South Atlantic right whales). We are measuring contaminant levels in both populations and using cell lines to determine which pollutants pose the greatest risk.
|This graph shows chromium levels in North Atlantic right whale skin tissues.|
Metal analysis was made in skin biopsies sampled from live North Atlantic right whales. We tested 21 metals, including both essential and non-essential elements. This analysis showed us that the right whale have relatively high levels of certain heavy metals, such as chromium.
We are investigating the genetic effects of environmental contaminants on North Atlantic right whales by using cell lines developed from right whale skin, lung, and testis.
|Right whale skin cells
(10X microscope magnification)
In this picture, you can see the cells growing out from a skin biopsy (the black area).
|Right whale lung cells
(10X microscope magnification)
Our research focuses on five classes of environmental contaminants (polycyclic aromatic hydrocarbons, metals, anti-fouling agents, anticorrosives, and radionuclides) that pose a specific concern for right whales (1) and investigates the genetic effects of these chemicals on its cell lines.
We are also interested in investigating the genetic effects of environmental contaminants on North Atlantic right whales by using their closest living relative, the bowhead whale, as a surrogate model, since the bowhead whale is not endangered as the North Atlantic right whale.
With the data obtained from these efforts, we will be able to determine:
1) Which classes of contaminants damage the reproductive organs of whales;
2) The sensitivity of reproductive organs compared to somatic tissues;
3) How bowhead whale cells compare to right whale cells.
Furthermore, this proposal investigates cell lines from animals of different age classes (adult and sub-adult), different genders and multiple individuals from both species. This work will help us to begin to understand the intra-species differences (age, sex and individuals) in the response to the genetic effects of environmental contaminants by using cell lines from each animal. And also look at the differences between reproductive and somatic tissues.
In addition, we are interested in understanding the mechanisms of DNA damage repair in these species and compare with our human studies.
This study will greatly enhance our knowledge of the physiology and toxicology of the North Atlantic right whale. Moreover, it will create tools (cell lines) that can serve as right whale-specific models, which can be used by other investigators to better understand additional aspects of right 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 right 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).
|Right whale normal metaphase (100X microscope magnification)
(42 chromosomes, male individual)
|Examples of damaged chromosomes in right whale cells (100X microscope magnification)
Chromatid gap (red arrow)
We investigated the toxic effects of hexavalent chromium, a well know human carcinogen in three cell types, lung, skin and testes.
Chromium caused cell death in all cell types, being the testes the most sensitive cell type.
|This graph shows that soluble chromium (VI) is cytotoxic to 3 different right whale cell types.|
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 3 different right whale cell types.|
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 nuclei 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:
Pictures coming soon!
- 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 vinicity 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):
|Right whale skin cell nuclei with no DNA double strand breaks.|
|Right whale skin nuclei with several DNA double strand breaks.|
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 right 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 right 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 right 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.
|Right whale lung cells expressing GFP (under normal light)|
|Right whale lung cells expressing GFP (under fluorescent light)|
(Soon to come!)
Kraus, S.D. and Rolland, R.M. (2007). Right whales in the urban ocean. In The urban whale: North Atlantic right whale at the crossroads (Kraus, S.D. and Rolland, R.M., Eds), pp. 543. Harvard University Press, Cambridge, Massachusetts.
Reeves, R.R., Rolland, R., Clapham, P.J. (eds) Causes of Reproductive Failure in North Atlantic Right Whales: New Avenues of Research. Report of a Workshop Held 26-28, April 2000 p10-11.
Li Chen, T., Wise, S.S., Holmes, A., Shaffiey, F., Wise, Jr., J.P., Thompson, W.D., Kraus, S. and Wise, Sr., J.P. A Comparison of the Genotoxicity of Hexavalent Chromium in Human and North Atlantic Right Whale (Eubalaena glacialis) Lung Cells. Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology, 150(4): 487-494, 2009.
Li Chen, T., Wise, S.S., Kraus, S., Shaffiey, F., Grau, M., Thompson, W.D., Zheng, T., Zhang, Y., Romano, T., O’Hara, T. and Wise, Sr., J.P. Particulate Hexavalent Chromium Is Cytotoxic and Genotoxic to the North Atlantic Right Whale (Eubalaena glacialis) Lung and Skin Fibroblasts. Environmental and Molecular Mutagenesis. 50:387-393, 2009.
Ierardia, J.L., Manciab, A., McMillan, J., Lundqvist, M.L., Romano, T.A., Wise, Sr., J.P., Plant, A. and Warr, G.W. Sampling the Skin Transcriptome of the North Atlantic Right Whale. Comparative Biochemistry and Physiology.4: 154–158, 2009.
Wise, Sr. J.P., Wise, S.S, Kraus, S. Shaffiey, F., Grau, M., Li Chen, T., Perkins, C., Thompson, W.D., Zheng, T., Zhang, Y., Romano, T., and O’Hara, T. Hexavalent Chromium Is Cytotoxic and Genotoxic to the North Atlantic Right Whale (Eubalaena glacialis) Lung and Testes Fibroblasts. Mutation Research. 650: 30-38, 2008.
Godard, C.A.J., Wise, S.S., Kelly, R.S., Goodale, B., Kraus, S., Romano, T., O’Hara, T., and Wise, Sr., J.P. Benzo[a]pyrene Cytotoxicity in Right Whale (Eubalaena glacialis) Skin, Testis and Lung Cell Lines. Marine Environmental Research 62:S20-S24, 2006.
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. 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 Southern Right whale samples.
Dr. Scott Kraus is Vice President of research at the New England Aquarium. He also operates the research field station in Lubec, Maine. He provides expert advice and guidance on North Atlantic right whales and access to samples. He is also collaborating with Dr. Wise on a project investigating the toxicity of metals to lobsters.
Dr. Tracy Romano is the Vice President of Research and Veterinary Services, Mystic Aquarium & Institute for Exploration. She provides expert advice and guidance on the immune system in marine mammals.
Dr. Todd O'Hara is a researcher with the Institute of Arctic Biology at the University of Alaska, Fairbanks. He provides expert advice and guidance on bowhead whales and helps with access to the field station in Barrow, Alaska.
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. Mariano Sironi is the scientific director of the Instituto de Conservacion de Ballenas (Whale Conservation Institute) in Patagonia, Argentina. He is part of the effort to study the Southern Right whales.
Dr. Vicky Rowntree is Research Associate Professor of Biology, University of Utah, and co-founder of the Ocean Alliance. She provides expert advice and guidance on Southern right whales and access to samples.
Dr. Douglas Thompson is a Professor of Epidemiology and Associate Director of the Maine Center for Toxicology and Environmental Health at the University of Southern Maine. He provides expert advice and guidance on statistical analysis and study design and also assists with the marine mammal studies.
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 was generously supported by grant number NA03NMF4720478, "The Genetic Effects of Environmental Contaminants on the Reproductive Systems of the North Atlantic Right Whale (Eubalaena glacialis)" from the National Marine Fisheries Service of the National Oceanic and Atmospheric Administration and by the Maine Center for Toxicology and Environmental Health.