Cells Between Life and Death

What do you get if you mix a pharmacologist - a drug specialist - with a chemist? Well if you are Murdoch scientists Garth Maker and Ian Mullaney then the answer is a totally new way of profiling how cells respond to toxic - or therapeutic - substances.

They've been profiling all of the chemicals made by cells - both when they are healthy and when they are injured - in order to develop a way to find new drugs that can reverse the damage done by a stroke. But their system has also enabled them to look at pesticide chemicals we used to think were safe...

Ian - Well, one of the problems that's been interesting me for a long time is, what happens when cells get injured. It's clear that when cells get injured either by some sort of mechanical trauma or chemical, or rather exogenous trauma, the cells won't immediately die. They're go into a period of being sick and then into a protective mode. In stroke for example, the stroke infarct, the area of dead tissue, a round about that becomes an area called the penumbra which are cells which are susceptible to damage. What we think there is there's a window of opportunity for example to actually protect those cells.

Chris - So, there is this period after a cell is injured when it maybe destined to die if there's no intervention, but not necessarily. And if you can work out what's going on when it goes into this mode, upstream of dying, you may be able to turn the tables and reverse the death process.

Ian - That's right. What seems to happen is that the cell starts to release compounds when they've been compromised. One of the major ones that they releases is a compound called glutamate and glutamate is ultimately a cell killer. But it seems that in these early stages, the glutamate is actually acting as a protector.

Chris - Garth, you're trying to do this by looking at what chemicals are in cells and come out of cells during these injury processes and the recovery processes. How are you actually doing that?

Garth - The basic principle of metabolomic analysis is that we want to profile as many of these small molecule metabolites - things like amino acids, sugars, fatty acids in a single sample as we can. So, we take the cells, we extract the metabolites, and we then use a combination of chromatography and mass spectrometry techniques in order to profile not only identifying which compounds are present in the sample. But also, how the levels change from one sample to another, which we can then use to actually see which changes are occurring in a specific sample that are due to the treatment or due to the toxic event.

Chris - So, Ian hands you some cells which he has made sick and you can then put the cells through your analytical process and come up with a profile. Not just at that moment in time, but I presume you could look at different points in time to work out how processes biochemically are changing around these cells as they go through this injury process.

Garth - Absolutely and that's one of the key advantages of the technology is  that we get these very large amounts of data that tell us exactly what's going on in a whole range of biochemical pathways at both multiple time points and also, over a range of doses as well. So, we can get a true picture of the pharmacology or the toxicology of these compounds.

Chris - So, what is this is actually showing you, Ian?

Ian -  What we're able to do is to take that data and then we can look at the data produced from the healthy cells and then compare it to the data produced from the cells that have been insulted. What we then actually end up with is actually a pictorial representation of the data. It makes it very easy to see the changes then. In the graft that we've produced, we can see a little hot spot of important chemicals which give us a normal condition and then we can see a shift in the graft. By looking at the shift, that gives us an indication of the damage and then we can maybe try and reverse that damage by adding protective drugs, to see if we can take the shifted pattern back to the normal pattern.

Chris - So, it's not just a question of saying, "Can we look for drugs that will stop this happening?" But you could also ask, "Why do some drugs cause damage? Or even, do they cause damage?" Because if you've got a new drug no one has ever seen before, you could put it into that kind of assay and see if the cells produce a metabolic profile in Garth's tests that are similar to the ones that are produced by known toxic agents. If they do, you know immediately this new drug may be harmful.

Ian - That's right. We've got quite a load of data now looking at methamphetamine and we can actually show when we treat the cells with methamphetamine, we can see these characteristic shifts from the well cell pattern to the sick cell pattern. And that could be extended to other drug of abuse and that's something that we're interested in at the moment anyway.

Chris - Obviously, if you can work out why some things cause bad things to happen, that may give you an insight into how to make good thing happen.

Ian - Yes, that's right. One of the things that again, we're interested in as actually screening compounds as neuroprotectants. What we have is a number of compounds planned to investigate. We make the cell sick. We can look at the pattern of the sick cells and then try and reverse that to make the cells healthy again.

Chris - Sounds so obvious. Now we're all having the sort of conversation doesn't it, to think you put a chemist like you Garth with a pharmacologist like you Ian, and you can do this. Why is no one doing this?

Garth - The technology for metabolomics is really only taken off in the last decade. I think it's the beautiful thing that we get in science which is where we have people with different skills coming together and realising that actually, we can combine these new skills and new technologies in exciting ways to look at things that we haven't been able to previously do.

Chris - And is it just drugs Ian or could we look at other things as well? People are quite worried about environmental contamination, that kind of thing.

Ian - No. Of course, we can actually look at any factor which has caused damage in these cells. So for example, we have a number of projects looking at pesticides - short term and long term exposure and we find changes in the metabolite profile of these cells which mimic normal damage that we've seen in cells.

Chris - Dare I ask, which insecticides you're seeing damage from?

Garth - So far, we've focused on ones that people will be commonly exposed to, primarily Permethrin which is found in a lot of flea treatments for household pets and also Malathion which is found primarily in head lice treatments. And we see not only do we have biochemical effects at very high concentrations as you might see during an accident, an exposure, but also chronic low level effects, and effects where we see a single one off event that then has a lasting effect within the cell over an extended period of time.

Chris - These compounds that are in common use because we regard them as safe. You're saying that in your assays, you can detect changes in cells biochemically which mirror changes caused by known very bad toxins.

Garth - And obviously, there's going to be a threshold whereby this will see some low level biochemical effects which are not in the long term toxic. At certain levels of exposure over certain periods of time, we will pass that point and we will see long term toxic exposure due to these compounds and that's something that I think we don't have enough data as to the long term effects of these compounds which are so prevalent in our environment.

Chris - So, what are the implications then for these things that are ubiquitously used in the environment - the coatings on the insides of the tin can that baked beans come out of? There must be all kinds of chemicals that this sort of level of scrutiny has never been done for.

Ian - That's absolutely, right. I think one of the problems that I think they've faced in the past is that we haven't been able to detect the changes at the low levels that these compounds are present in these products. And it's only now with the technologies, the mass spectrometry in particular that we can actually find effects caused by varied levels of exposure to these compounds.

Chris - Obviously, we've talked about brain cells a lot, but if you could probably do this for any bit of the body, couldn't you Garth?

Garth - Absolutely and our next focus is going to be on liver cells since these are the cells that are primarily going to deal with a lot of these toxic or potentially toxic compounds as they enter the body and indeed any other cell type that we are interested in, we could culture the cells and look at the effects of them using the same system, absolutely.