A menace to mussels: Illicit drugs in waterways induce oxidative stress

Drugs in river water

Illicit drugs and their metabolites do not affect drug users alone, they also affect the wildlife in rivers and seas because residual drugs are excreted into the water system and are removed inefficiently by wastewater treatment plants. The discovery of drugs of abuse in the river Po in Italy in 2005 led to several different studies in which the aquatic levels were used to determine the level of use in the local catchment areas.

One of the most abundant drug-related compounds that has been found regularly is benzoylecgonine (BE), the primary metabolite of cocaine. Yet, despite its common occurrence at levels as high as 3 µg/L in wastewater and 520 ng/L in surface water, there have been few studies on the effects of BE on the local aquatic life.

Now, European scientists have examined the effects that BE might have on the freshwater mussel, Dreissena polymorpha, using a proteomics approach. This organism is widespread throughout Europe and North America so is a popular choice for exotoxicological studies. Alessandra Pedriali and colleagues from the University of Milan, ISB Ion Source & Biotechnologies S.r.l., Gerenzano, and University College Cork examined the effects of oxidative stress, which is a common effect of environmental pollutants on freshwater species.

Reactive oxygen species are produced as a result of contamination and these can cause structural changes in proteins. For instance, the lysine, arginine and proline residues in proteins are susceptible to oxidation to carbonyl groups while thiol groups in cysteine and methionine residues can be oxidised to sulphenic, sulphinic or sulphonic acid. All of these structural changes are capable of inactivating proteins or stimulating their breakdown, affecting the way an organism functions.

Fluorescent labels for protein modifications

The way to detect protein thiols and carbonyl groups is to label them with fluorescent reagents which aid their detection on electrophoresis gels. The researchers gathered colonised rocks of the freshwater mussels from an Italian lake and transferred them to tanks in a lab where they were maintained and fed. They were exposed to a constant concentration of BE over 14 days, then their gills were removed and the proteins extracted.

The protein thiol groups were labelled with 5’-iodoacetamide-fluorescein which does not attach itself to the different types of oxidised thiols. So, a comparison of control mussels with BE-treated mussels will be able to detect any changes in thiol group concentrations. The carbonyl groups were reacted with fluorescein-5’-thiosemicarbazide.

All of the proteins were separated by one-dimensional gel electrophoresis and the fluorescent tags were analysed to enable the levels of proteins to be estimated. Then two-dimensional gel electrophoresis was also employed and the proteins which had different abundances between the controls and those treated with BE were identified by mass spectrometric methods.

Indicators of oxidative stress

The effects of BE on the thiol groups in the gill proteins appeared to be quite small because the total thiol content measured after 1D gel electrophoresis remained unchanged after treatment. However, there was a significant increase in the number of carbonyl groups over the same test period.

A total of 20 protein spots were selected from the comparative 2D gels for protein identification. There were other candidates but they were regarded as too low in abundance or too close to other proteins on the gel for clean isolation and analysis. The candidates included thiol-containing proteins as well as carbonylated ones. Of these, only eight could be identified and the failure to identify the others successfully was attributed to insufficient tryptic peptides or low database matching scores.

Two of the affected proteins were a heat shock protein, which suffered thiol oxidation, and cytochrome c, which became more carbonylated after BE exposure. Since both of these proteins are generally associated with oxidative stress, it supports the notion that the freshwater mussels were undergoing the same stress. This conclusion is also consistent with published data on equine cytochrome c, identifying BE as a pro-oxidant.

Three of the other affected proteins were different forms of tubulin which experienced reduced carbonylation after BE treatment. This behaviour might be due to glutathionylation, which is induced to allow the organisms to survive high levels of oxidative stress, but more work is needed to confirm this hypothesis.

Three enzymes involved in energy metabolism, namely the glycolysis pathway or oxidoreductase activity, were also modified, giving further support to changes induced by oxidative stress.

Although the researchers only managed to identify a few of the proteins affected by exposure to BE, they are consistent with the idea that BE induced oxidative stress in the freshwater mussel. This conclusion could be supported by further studies investigating the changes in protein abundances after different BE doses, which might also reveal the mechanism of action of oxidative stress to tell us exactly how drugs of abuse in our waterways affect the wildlife living there.