Feature: Free radical

This feature appeared in the November/December 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.

In her youth in Germany, Uta Wille always imagined herself becoming a biologist. However, after being exposed to the joys of chemistry in her undergraduate years, she found herself bound to the more fundamental nature of this science.

After graduating with an undergraduate degree in chemistry, she went on to complete a PhD at the University of Kiel on atmospheric chemistry and free radicals, followed a few years later with a habilitation. For this, Wille shifted her research focus from physical to organic chemistry and, in the process, rediscovered her inner biologist.

“After a post-doctoral stint in Switzerland with a group looking at free radicals and DNA, I started to wonder what all these atmospheric radicals that I had worked on for many years actually do to biological systems such as cells and tissues,” she says.

“Nobody had ever looked into this, mainly because atmospheric chemists only talk about chemistry to other atmospheric chemists. They don’t talk to biochemists, and vice versa of course. So I became fascinated with the missing link between these two different areas . It was an unmined niche.”

Wille moved to from Germany to Australia in 2003 to pursue her fascination and is now a chief Investigator within the ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, located at the Bio21 Institute in Melbourne.

Loose cannons

In chemical terms, radicals (often called free radicals) are atoms or molecules with an unpaired electron, which makes them highly chemically reactive. These molecules are everywhere in nature and play important roles in combustion, atmospheric chemistry, polymer chemistry and many other chemical processes, including biochemistry and human physiology.

Although necessary for normal function, free radicals in biological systems are known to also induce cell damage. Over the years they have been cast as the villains in various aspects of human physiology including ageing, degenerative disease and cancer.

According to Wille, radicals from atmospheric pollutants and UV radiation can also cause biological stress. “As part of the centre’s research program we and others here in Melbourne are interested in the mechanisms by which environmental radicals and UV light from sunlight damage DNA and cellular systems, and in the different repair mechanisms induced in biology to deal with that damage,” Wille says.

It is well established that exposure of skin to UV light can ultimately result in cancer due to a chemical reaction with the skin cell DNA involving the formation of pyrimidine dimers.

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The normal DNA repair process that goes on in humans is designed to clip out any damaged DNA before replication, so that all proceeds as normal. However, this dimer formation caused by free radical exposure may introduce DNA mutations such that the damage is sometimes not correctly recognised and repaired, leading to disease.

Interestingly, many other species including marsupials, some plants and also certain bacteria use a different repair system from humans in response to free radical damage. In such cases, the presence of sunlight also converts the damage-causing pyrimidine dimers to their harmless monomeric form.

This happens when a light-sensitive DNA ligase enzyme present in these repair systems catalyses electron transfer to facilitate repair of the damaged DNA. However, despite many hours spent by many scientists, the details of this light-harvesting repair mechanism – how it works from planting the unpaired electron onto the system until the DNA molecule is restored – remains unknown.

Accordingly to Wille, controversy reigns and many chemists have worked hard to identify every single chemical step in the process, without much joy.

“The problem was that nobody has ever been able to see intermediates in this dimer cleavage process. Computational studies contradicted experimental studies and no consensus was reached as to exactly how this thing was happening.”

But now, Wille together with her collaborator in Innsbruck, Linda Feketeová, have for the first time detected chemical intermediates as well as other byproducts produced during the enzyme-cleavage DNA repair process.

“This showed finally that it is not a clean mechanism and other processes are going on that we have to also understand as part of the whole scheme,” says Wille.

The team achieved their success by using a very sophisticated version of gas-phase mass spectrometry developed by their physicist colleagues in Innsbruck. “These guys set up a model system of simulated damage whereby they could actually plant electrons with a defined energy into the process and follow them as the reaction proceeded.

So, although we do not look at DNA, we look at the chemical damage itself or, more precisely, the structural motif of the damage, which we can synthesise in our lab. We have used this system of analysis for many other things in the past in our studies of environmental radicals.”

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Facilitating repair

Identifying such chemical intermediates of radical-induced reactions in biological tissues is fundamentally important for understanding the mechanisms of DNA repair that happen consequent to the process.

Indeed, when this work was published by Wille and Feketeová last year, it gained a lot of attention due to the promise of using these findings for developing a novel way to block skin damage from sunlight-derived UV light. In other words, a new and better sunscreen!

“So, this enzyme from non-human systems does indeed have potential use biologically in humans, such as in agents to repair UV damage to skin and thus prevent skin cancer,” Wille says.

“In fact, some people have now shown that topical application of this enzyme reduced formation of the pyrimidine dimers in skin cells, thus reducing the radical-induced cleavage and associated DNA damage.”

However, Wille cautions that before such an agent can be transformed into an approved commercial reality as a miracle sunscreen, scientists like her still have plenty to do in understanding the precise details of the chemistry involved.

“We can’t put something on our skin if we don’t really understand all the biochemical implications. We know that repair occurs, but then we also see byproducts and we just don’t know if these are all safe.”

On a brighter note, thanks to scientists like Wille bridging the biologist-to-chemist divide, we now have better techniques to look at such processes that are set off in all of us by these environmental free radicals.

According to Wille, it is all down to “the very specialised mass spectrometer instruments that the physicists in Austria make and then fiddle with to give us just the information we need. These guys are definitely the handymen of the science world. They sit there with their screwdrivers and welding kits and just make it happen!

The secret was working out how to adjust the energy of the electron being attached down to virtually nothing. The fact that they think of these things is just fascinating. It also might explain why past experiments never revealed the reaction intermediates despite people thinking they must be there. They were there, but without using zero energy, people just could not find them.”

Dr Uta Wille is a chief investigator in the ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, and senior lecturer in the School of Chemistry at The University of Melbourne.

Wille has been a pioneer in combining physical and organic chemistry in the study of free radicals. Her current research program targets the chemistry of reactive intermediates formed by merging radicals of atmospheric importance with biological systems.

Her group also studies the role that certain atmospherically important radicals play in diseases of the respiratory tract through oxidative damage of biological molecules that are in direct contact with the environment. Their work suggests that nitrate radicals in atmospheric pollutants could be the real culprit in some pollution-derived diseases including asthma.

This feature appeared in the November/December 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.