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Wisconsin Researchers Use Proteomics to Hunt Biomarkers Tied to Radiation Damage

Name: Mukut Sharma
Position: Associate Professor, department of medicine, Medical College of Wisconsin, 2006 to present
Background: Senior research associate, department of medicine, Kansas University Medical Center, 1990 to 1994; director R&D and market planning, Ira Lab Private, Baroda, India, 1987 to 1990; postdoctoral, neurobiology, Ohio State University, University of California, Los Angeles, 1980 to 1981; MSc, PhD, biochemistry, University of Jodhpur, India, 1971 to 1979
Despite the ongoing risk of radioactive dirty bombs set off by terrorists, as well as run-of-the-mill nuclear disasters, there are currently no protein biomarkers that could help detect early signs of injury brought on by radiation exposure.
With this in mind, researchers at the Medical College of Wisconsin have been studying changes in rat protein levels after radiation exposure, and in an article published in the June 18 online edition of Proteomics – Clinical Applications, they used proteomic technology to detect those changes in rat urine.  
Writing in the paper, the authors describe the potential of using those changes to help identify biomarkers associalted with radiation damage.
Among their findings are 188 proteins that increased in abundance by at least two-fold 24 hours after the rats were exposed to radiation and 76 proteins that decreased in abundance by at least two-fold.
However, the researchers cautioned that it’s too early to say they have identified any candidate markers.
This week, ProteoMonitor spoke with Mukut Sharma, senior author of the article, about the research. Below is an edited version of the conversation.

Talk about the need for these kinds of biomarkers. Aren’t there ways already to detect radiation exposure and then to figure out the probability of radiation injury?
As a matter of fact, there are no known markers of radiation injury. Now, there are radiation injuries that can occur in several ways. The most common given source of radiation is X-ray that we receive during therapeutics. And the next is radiation therapy in which we receive actually substantial amounts of radiation.
The other kinds of radiation doses that we are likely to get, ionizing radiation, are only because of some accident or spillover, so [we’re talking about] some kind of a dirty bomb.
Keeping those first two therapeutic radiations in mind, there are no known human models that can be studied really to anticipate what kind of dose and what kind of injury one will receive. In therapeutic radiations, X-rays give such miniscule amounts of radiation that you’ll have to get about 10,000 X-ray exposures before you get any substantial amounts of radiation.
In terms of therapeutic radiation, cancer therapy primarily, what is done is [exposing] the body or part of the body to basically neutralize the immune system. Those are harmful enough, and many of those people, about 20 to 25 percent … will end up developing some type of chronic disease, primarily kidney disease.
Over time, people have reduced total body radiation prior to bone marrow transplants and things of that kind. But still it’s a widely accepted therapeutic practice. Newer practices include immunoligand kind of therapies in which radiation sources tied to an antibody are targeted to a particular organ or cell type.
A majority of these types of radiation therapies also end up harming the kidneys because these antibodies circulating these products end up in the kidney, and over time they injure the kidney.
Then came in 2001 [the Sept. 11 terrorist attacks in New York City and Washington, DC], this certain kind of realization that we could be exposed to radiation, and that kind of opened up the realization that in cities such as New York … thousands of people could get doses of radiation, which are unmeasured and that could be basically [either] purely harmless [or] lethal.
If the radiation doses are non-lethal, it’s hard to imagine what kind of radiation one has received, and that’s why Congress …in 2004 … instituted a program called Bioshield. It was a Congress-mandated program that required preparedness for radiation attacks. Under that program … regional centers were established … to find out therapeutic agents that would protect against radiation injuries.
But the question that we faced … is how to measure this injury. And we know that there is no definitive measure or marker of early injury. I’m emphasizing early versus late because as the radiation injury progresses, the body’s response and organ failures occur over time.
In humans, going back to … Nagasaki, low-dose exposure to radiation took several years for people to develop cardiovascular problems, about 10, 20, 30 years later. Some of these people are still alive, and there was a joint study between the Japanese government and the US government that continues to date.
And there was a large number of cardiovascular complications detected in people who apparently were thought not [to have received] any radiation because there was no measure of radiation. So they raised the possibility that there could be non-lethal exposures, yet over time, these people might develop complications.
This is the situation we have with rat models, to study this particular kind of condition. … People who had been working with this rat model did not think there were any early signs of radiation injury, that disease certainly appeared in these rats at about six weeks after total body radiation, and the rats would progressively deteriorate and die about six months or nine months [later].
And this particular timeline parallels human conditions. People who received total body radiation in therapeutic regimens, many of them, like 20, 30 percent end up developing kidney problems or other cardiovascular problems, hypertension especially, two to three years, or even later, after the disease therapy was over.
Does Bioshield have a component for biomarker discovery?
Most of these programs that were instituted were with a specific mission to find therapies or mitigation agents, but under each of these programs were certain pilot grant programs. I’m a part of one of the major center grants for therapeutic agent discovery, but I also applied for a pilot grant … for marker discovery, proteins being my long-term interest.
I looked at the urine proteome and, of course, I knew from my background that one, there was a lack of such biomarkers, which could be detected early on, and two, kidney is a good target tissue because it represents deep-tissue injury. And urine, because of the proteome composition, is simpler compared to plasma by several orders of magnitude.
On the other hand, while urine is a filtrate of plasma, it is also carrying the directly derived proteins from kidneys as well as the urinary tract, so it represents deep-tissue injury as well as superficial injury.
Where are we in terms of treating radiation exposure? Even if you are successful in finding these markers, can we somehow reverse the damage done by radiation, or at least treat it?
There are people … who are looking at a combination of drugs as well as compounds that are … off-label to look at their applications in terms of mitigation. The idea is to catch or identify people who have received relatively low doses of radiation and start immediately with a combination of agents.
The sticky point is that we don’t know who has been exposed and who has received how much radiation. And in that regard, biomarkers come in handy.
There are several kind of preliminary studies, none conclusive, to initiate mitigation. Our center, for example, has been working long-term on ACE inhibitors and receptor blockers. They have been working on chronic injury caused by radiation.
And we have a rat model [where we have achieved] a fair amount of success. Other groups … have been looking at alternatives of kinase inhibitors, for example.
But there is a lot of emphasis going on through this program, mostly on medication and treatment.
Which were the more interesting proteins that you found, the ones that increased in abundance or those that decreased in abundance?
I don’t think I can give priority [to one or the other]. I would be happy if we can find something in terms of proteins that increase because they are easier in terms of developing tests and identification markers and diagnostics. But I would not put a penny on any of these things right now because it could go either way.
Of the proteins that increased in abundance, is it clear to you that this is happening because the body is trying to repair itself after radiation exposure? Or why are they increasing?
It could happen both ways. One, they have lost their bearings in terms of their organization in the body, and two, they are trying to kind of dissociate and fall apart, basically.
What comes in response is at a later time point, and probably we’ll have to end up looking at a comparison between plasma as well as urine tissue and see what happens in both of them.
Of those proteins whose abundance increased, have you identified any that are especially interesting for follow-up?
As you know, this radiation injury results in proteinuria by kidney failure — in people as well as in rats — and hypertension. Both these conditions cause severe loss of protein in the urine, but these things appear, let’s say, six weeks down the road in rats, and about a year or two after in people.
We found surprisingly … that within the first 24 hours [after radiation exposure] there is a drop in the urinary albumin, whereas in six weeks albumin is a predominant protein in urine. By itself … one could take this as an indicator, because there are very few conditions unless there is acute renal failure … where urine drops all levels of proteins [and] certain proteins specifically.
So that’s an interesting point to look at, to see what’s happening. And that could be taken up as an indicator.
At the same time, proteins that are increasing … one needs to follow up in time portions to see if they respond with time in terms of whether they disappear or continue to increase.
You’ve identified some changes that occur after total body irradiation. Is the next step then to research the significance of these changes?
One is [to determine] the significance, but even before that, to establish that there is some kind of a trend. … Even before we go for some kind of a larger study, we would like to see if at least one or two more species can be utilized to confirm this part. It should be a higher species than rat. That is always a stumbling block.
Are there human tissue samples from Chernobyl that you can study retrospectively?
We have applied for that, we requested [samples]. There is some work going and we were promised that when the time comes, we will be contacted or that we will be able to apply for this material.
But I’m aware of the work going on in the area, and we have tried to keep track, but the difficult part is [that] in pilot projects, the funding runs out before your ideas do. So, by the time you collect your team and you train people and you make contacts, these funds run out.
What’s the advantage of protein biomarkers for your work versus other kinds of biomarkers?
I look at protein biomarkers in two different ways. ... They could be specific down to the level of cell organelle compartment types. Number two, it is easier to develop diagnostics, for example, an antibody-based diagnostic that could be easily accessible and more economical in the long-term, if you consider storage for masses tens of thousands of people.
They last longer and are cheaper than anything I can imagine.
Smaller molecules, like metabolites of different kinds, while they are great, the major difficulty I feel is the non-specificity of their generation. [For] genomic applications, the difficulty … is the field applications at a larger level in terms of scaling up the application to bring it to every household.
I don’t know if [you can do that].
Is that the goal or vision that you have, that it would be a home-based diagnostic, versus a lab-based?
My dream is that we can develop a test that can be done at home. The reason is this: [If] a dirty bomb [goes off] in a crowded area, the likelihood of people getting affected is in a very narrow area, the immediate vicinity.
But a large amount of radiation will flow into air, and depending on the direction and density of the air flow, it will affect two different levels. And it’s not supposed to go very, very far … but the news of such an event will go much further than these particles, and I believe that tens of millions of people will consider themselves having been exposed to radiation.
So the hysteria and mass fear [that would accompany] this type of event is likely to be much larger than the number of actual people who will receive damaging doses of radiation.
If I can sit at home and receive peace of mind thinking, ‘OK I don’t seem to be affected, I don’t have to go to the doctor tomorrow morning,’ that will offload the burden off the healthcare professional who will have a lot to do at that time.
So if people like me don’t show up, or I can triage myself at home and not bother the healthcare industry, that will be a lot of relief to society.

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