CONTAMINANT SUSCEPTIBILITY GENES IN NORTH ATLANTIC RIGHT WHALES

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Lee Carroll
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1 North Atlantic Right Whale Forum - Mark Hahn CONTAMINANT SUSCEPTIBILITY GENES IN NORTH ATLANTIC RIGHT WHALES MARK HAHN, WOODS HOLE OCEANOGRAPHIC INSTITUTION. Hahn studies molecular interactions of pollutants and marine organisms.» Biography» PDF version of talk (232 kb) My laboratory has just recently entered the arena of right whale research. I will borrow a phrase from Doug Nowacek: This talk is going to be relatively uncontaminated by data. Instead, what I d like to do is to present more of a conceptual talk, to highlight: some of the problems and challenges that we face in trying to understand the impact of contaminants on the right whale some of the data gaps potential approaches that might fill those data gaps The value of forums such as this is that they bring together people from diverse backgrounds to attack a common set of problems. My background is in biochemical and molecular toxicology. I think in terms of molecular mechanisms, things that are going on at the level of the cell and below. Understanding those mechanisms can perhaps help us understand the impact of contaminants on animals that are exposed to them. What is the role, if any, of contaminants in the low reproductive success of these right whales? Let me begin with a few general things we might consider in thinking about this question. First, there are parallels in the way we look at the effects of contaminants on marine mammals, and on human health. One parallel is the difficulty of direct experimentation and sampling in either humans or marine mammals. Another is the separation in time of exposure and effects: In both humans and whales, the life stages

2 most sensitive to contaminant exposure occur just before or just after birth, but the effects might not be seen until adulthood. An effect seen in an adult female may in fact be due to contaminants passed to her in her mother s milk. This fact makes it very difficult to do the kind of epidemiological studies in humans and marine mammals that can implicate a particular contaminant in a particular effect. Second, for the chemicals I m interested in studying in right whales, there is a large database of studies in laboratory animals, over a number of years, on the effects of contaminants and their dose dependence. The National Institutes of Health pours hundreds of millions of dollars into this work, and so there s a lot of information. The question is: Can we use this information to learn about contaminant effects in right whales? It would be nice if we could take a piece of blubber or a copepod and send it out for chemical analysis and say, Measure everything that s in there. But you can t find something if you don t look for it. If you don t tell labs to look for a particular chemical, they won t find it. The same is true for effects: What kinds of effects should you look for in whales? Finally, our lack of very basic information on the normal physiology, biochemistry, and endocrinology of right whales really limits our ability to understand what might be abnormal in these animals. That s why there s a real need for the kind of basic studies that Rosalind Rolland, Michael Moore, and others are doing. Ultimately, we re interested in the impact of contaminants on the entire population of right whales. But we really need to begin by understanding the impact on individuals. The procedure that s traditionally used in human and ecological health studies is called ecological risk assessment. It involves two components. The first is exposure assessment, or figuring out how much of the contaminant the animals are actually exposed to, and when they re exposed to it. The second is hazard assessment, which means defining the effects of contaminants, and then looking quantitatively at the relationship of those effects to the doses administered. Putting those two factors together, you can come up with a probability that an individual animal would be affected by a particular chemical at a given exposure level. On the exposure side, what do we know? Michael Moore talked about the work that he and others have done, measuring levels of some contaminants both in right whale blubber and the animal s food source, copepods.

3 TThese include PCBs (polychlorinated biphenyls), PAHs (polyaromatic hydrocarbons), and organochlorine pesticides. What we don t know are the presence and effects in right whales and their food of other chemicals, including chlorinated dibenzodioxins and dibenzofurans and emerging contaminants (meaning pollutants that haven t been studied until very recently), such as polybrominated diphenyl ethers, tetrabromo bisphenol-a, and several others. The important thing about the brominated contaminants is that they are still being used. They haven t been banned in thiws country yet, though they may have been banned in Europe. Phthlates and perfluoro-octane sulfonate, better known as Scotchgard (3M), are other types of emerging contaminants that people are starting to study. Another question is the role of PCB metabolites, such as hydroxypcbs. These contaminants aren t necessarily present in the food whales are eating, but they re produced by whales when they bio-transform the PCBs they ve absorbed. Once metabolized, these products may act by completely different mechanisms than the parent compounds. Thus, we need to know something about how whales are metabolizing parent compounds into products that might be more toxic. We really need to get a handle on the exposure of right whales to all these compounds. We need to look at the whales food source, blubber, and tissues, when we can get them. If it were possible to get some idea of the concentrations of such compounds in whale milk, it would help us assess exposure in the critical time right after birth. This issue is further complicated by the fact that many contaminants are mixtures of compounds. Look at the dioxin-like compounds, so called because they act by the same mechanism as dioxin. Dioxin (2,3,7,8-TCDD) is what was in Agent Orange, used in Vietnam. Dioxin-like compounds include 75 different kinds of dioxins, 135 dibenzofurans, and 209 PCBs. Each has a different fate in the animal and different effects. How do we get a handle on this, or any suite of compounds that might be acting through any particular mechanism? One thing we might do is use something called a mechanism-based bioassay. This is an assay that doesn t look at individual compounds. Instead, it integrates all the compounds that act in a single way, by a single mechanism, and reflects the total biological activity of that entire mixture of compounds. For example, there is a liver-cell line that was produced by a collaborator of ours, Mike Denison from the University of California at Davis. These cells have had the gene for the green fluorescent protein integrated into them in such a way that, when they are exposed to anything that acts by a dioxin-like mechanism, they ll respond by increasing the synthesis and amount of this protein, which releases light as fluorescence. This response is something you can measure very easily and is directly related to the amount of dioxin-like activity in a mixture. We can use a variety of these mechanism-based assays, each targeting a different potential mechanism. These

assays will complement, not replace, the chemical analysis, and tell us something more about the kinds of contaminant exposures right whales are encountering.

4 assays will complement, not replace, the chemical analysis, and tell us something more about the kinds of contaminant exposures right whales are encountering. You might use these assays, for example, to look at copepods and see what dioxin-like activity is present. Now let s look at hazard assessment. It s very difficult to quantify dose-response relationships in right whales, so we have to rely on that large database of laboratory research on other animals and extrapolate it to right whales. First, let s consider what some of the effects might be. Again, let s use the dioxin-like compounds as an example. There are plenty of data for rats, mice, monkeys, and other laboratory species, relating perinatal (in utero through weaning) exposure to dioxin-like compounds to the effects on reproductive function seen in those animals at maturity. There are some general effects, including mortality, some altered sexual behavior, and reduced sperm counts in males. One thing we see in lab animals is that perinatal exposure to extraordinarily low concentrations of dioxin create effects that can t bee seen until years after the exposure. How do we then extrapolate the effects seen in lab animals to right whales? The process of extrapolation is fraught with uncertainty. We have to make so many assumptions that even extrapolating from a rat to a mouse can be very difficult. The biggest source of uncertainty is probably the animal s sensitivity. When you extrapolate, you assume that rats and right whales have the exact same sensitivity to these chemicals; that is, their dose-response curves are essentially overlapping. How do we determine whether the sensitivity of right whales to dioxin can be compared to the sensitivity of a laboratory species? Obviously, we can t do this directly, so we have to use an indirect approach. In human toxicology, scientists look at something called susceptibility genes. These are genes whose products the proteins they encode are involved in mechanisms of toxicity or disease. By looking at the variation in susceptibility genes in individuals or ethnic groups, scientists begin to understand the mechanistic basis for differences in susceptibility to compounds or diseases. Similarly, we might be able to use susceptibility genes to compare sensitivities of whales and laboratory animals. One likely example of a susceptibility gene in right whales is the gene for the arylhydrocarbon (AH) receptor, which is a target for the dioxins, PCBs, and PAHs. We know from studies on other species that when these compounds enter the cell, they bind to the AH receptor; the strength of binding is related to the compound s toxicity. When they bind, the receptor becomes active and turns certain genes on or off, resulting in toxicity through mechanisms we don t yet clearly understand. Other examples of susceptibility genes are the estrogen and androgen receptors, which can be targeted by organochlorine pesticides. Another, transthyretin, a thyroid-hormone transport protein that can be disrupted by the hydroxy metabolites of PCBs.

We can thereby infer something about the sensitivity of right whales to these compounds, as compared to the better-known experimental models. As I said, we ve just begun this work in right whales.

5 One objective of our work is to characterize these susceptibility genes in the right whale. Then we can compare the properties of these genes in right whales to the properties that we know exist in genes of mice, humans, and rats. We can thereby infer something about the sensitivity of right whales to these compounds, as compared to the better-known experimental models. As I said, we ve just begun this work in right whales. Joy Lapseritis is a student in my lab who has been working on this. Joy has cloned a partial sequence for the right whale AH receptor. She also will be studying some of the other receptors that act as susceptibility genes. Previously, we ve applied this kind of approach to the beluga whale, where we don t know the sensitivity, but we d like to know it. Brenda Jenson in my lab cloned the AH receptor from a beluga whale and compared its properties to those of the mouse and human AH receptors. What she found is that the beluga AH receptor had very similar properties to the receptor from a dioxin-sensitive strain of mouse. My hope is that, by doing some of these mechanistic studies, we can reduce the uncertainty both in exposure assessment and hazard assessment. Then we can obtain some reasonable estimates of the risks to individuals. In turn, we hope we can take these effects in individuals and somehow incorporate them into population models, thus asking some interesting questions about whether contaminants are likely to have an impact at the population level. That s the ultimate goal. I m grateful for this opportunity to interact with all of you, and I m looking forward to further discussions. I d

6 also like to thank the Northeast Consortium, which really got us started on the right whale business a little while ago. Biography Mark Hahn earned his PhD in toxicology. His research on right whales has focused on the impact of chemical contaminants in the whale s low reproduction rate. Hahn conducts studies on the biochemistry and molecular biology of the receptors and enzymes involved in chemical-biological interactions.

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